Methods of treating conditions with antibodies that bind colony stimulating factor 1 receptor (CSF1R)

ABSTRACT

Methods of reducing cytokine levels and methods of treating conditions with antibodies that bind colony stimulating factor 1 receptor (CSF1R) are provided. Such methods include, but are not limited to, methods of treating inflammatory conditions, such as rheumatoid arthritis.

This application is a divisional of U.S. application Ser. No.14/014,446, filed Aug. 30, 2013, which claims the benefit of U.S.Provisional Application No. 61/695,641, filed Aug. 31, 2012; 61/767,989,filed Feb. 22, 2013; and 61/778,706, filed Mar. 13, 2013; each of whichis incorporated by reference herein in its entirety for any purpose.

TECHNICAL FIELD

Methods of treating conditions with antibodies that bind colonystimulating factor 1 receptor (CSF1R) are provided. Such methodsinclude, but are not limited to, methods of treating inflammatory andautoimmune conditions, such as rheumatoid arthritis, multiple sclerosis,and systemic lupus erythematosus.

BACKGROUND

Colony stimulating factor 1 receptor (referred to herein as CSF1R; alsoreferred to in the art as FMS, FIM2, C-FMS, M-CSF receptor, and CD115)is a single-pass transmembrane receptor with an N-terminal extracellulardomain (ECD) and a C-terminal intracellular domain with tyrosine kinaseactivity. Ligand binding of CSF1 or the interleukin 34 ligand (referredto herein as IL-34; Lin et al., Science 320: 807-11 (2008)) to CSF1Rleads to receptor dimerization, upregulation of CSF1R protein tyrosinekinase activity, phosphorylation of CSF1R tyrosine residues, anddownstream signaling events. Both CSF1 and IL-34 stimulate monocytesurvival, proliferation, and differentiation into macrophages, as wellas other monocytic cell lineages such as osteoclasts, dendritic cells,and microglia.

Many tumor cells have been found to secrete CSF1, which activatesmonocyte/macrophage cells through CSF1R. The level of CSF1 in tumors hasbeen shown to correlate with the level of tumor-associated macrophages(TAMs) in the tumor. Higher levels of TAMs have been found to correlatewith poorer patient prognoses. In addition, CSF1 has been found topromote tumor growth and progression to metastasis in, for example,human breast cancer xenografts in mice. See, e.g., Paulus et al., CancerRes. 66: 4349-56 (2006). Further, CSF1R plays a role in osteolytic bonedestruction in bone metastasis. See, e.g., Ohno et al., Mol. CancerTher. 5: 2634-43 (2006).

CSF1 and its receptor have also been found to be involved in variousinflammatory and autoimmune diseases. See, e.g., Hamilton, Nat. Rev. 8:533-44 (2008). For example, synovial endothelial cells from jointsafflicted with rheumatoid arthritis have been found to produce CSF1,suggesting a role for CSF1 and its receptor in the disease. BlockingCSF1R activity with an antibody results in positive clinical effects inmouse models of arthritis, including a reduction in the destruction ofbone and cartilage and a reduction in macrophage numbers. See, e.g.,Kitaura et al., J. Clin. Invest. 115: 3418-3427 (2005).

Mature differentiated myeloid lineage cells such as macrophages,microglial cells, and osteoclasts contribute to pathology of variousdiseases such as rheumatoid arthritis, multiple sclerosis and diseasesof bone loss. Differentiated myeloid lineage cells are derived fromperipheral blood monocyte intermediates. CSF1R stimulation contributesto development of monocytes from bone marrow precursors, to monocyteproliferation and survival, and to differentiation of peripheral bloodmonocytes into differentiated myeloid lineage cells such as macrophages,microglial cells, and osteoclasts. CSF1R stimulation thus contributes toproliferation, survival, activation, and maturation of differentiatedmyeloid lineage cells, and in the pathologic setting, CSF1R stimulationcontributes to the ability of differentiated myeloid lineage cells tomediate disease pathology.

SUMMARY

In some embodiments, methods of reducing the level of at least one, atleast two, at least three, or at least four, at least five, at leastsix, at least seven, at least eight, at least nine, or at least tenfactors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7,CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9 in a subject are provided.In some embodiments, a method comprises administering an effectiveamount of an antibody that binds colony stimulating factor 1 receptor(CSF1R) to the subject, wherein the antibody blocks binding of colonystimulating factor 1 (CSF1) to CSF1R and blocks binding of IL-34 toCSF1R. In some embodiments, the subject has an inflammatory condition.In some embodiments, the subject has a condition selected fromrheumatoid arthritis, juvenile idiopathic arthritis, Castleman'sdisease, psoriasis, psoriatic arthritis, ankylosing spondylitis, Crohn'sdisease, ulcerative colitis, lupus erythematosus, inflammatory boweldisease, inflammatory arthritis, and CD16+ disorders.

In some embodiments, the method comprises reducing the level of at leastone, at least two, at least three, or four factors selected from IL-6,IL-1β, TNF-α, and CXCL10. In some embodiments, the method comprisesreducing the level of IL-6. In some such embodiments, the subject has acondition selected from rheumatoid arthritis, juvenile idiopathicarthritis, and Castleman's disease. In some embodiments, the methodcomprises reducing the level of TNF-α. In some such embodiments, thesubject has a condition selected from rheumatoid arthritis, juvenileidiopathic arthritis, psoriasis, psoriatic arthritis, ankylosingspondylitis, Crohn's disease, and ulcerative colitis. In someembodiments, the method comprises reducing the level of IL-1β. In somesuch embodiments, the subject has a condition selected from rheumatoidarthritis and juvenile idiopathic arthritis. In some embodiments, themethod comprises reducing the level of CXCL10.

In some embodiments, the method comprises reducing the level of at leastone, at least two, at least three, or four factors selected from IL-6,IL-1β, TNF-α, and CXCL10. In some embodiments, the method comprisesreducing the level of IL-6; or the method comprises reducing the levelof TNF-α; or the method comprises reducing the level of IL-1β; or themethod comprises reducing the level of CXCL10; or the method comprisesreducing the levels of IL-6 and TNF-α; or the method comprises reducingthe levels of IL-6 and IL-1β; or the method comprises reducing thelevels of IL-6 and CXCL10; or the method comprises reducing the levelsof TNF-α and IL-1β; or the method comprises reducing the levels of TNF-αand CXCL10; or the method comprises reducing the levels of IL-1β andCXCL10; or the method comprises reducing the levels of IL-6, TNF-α, andIL-1β; or the method comprises reducing the levels of IL-6, TNF-α, andCXCL10; or the method comprises reducing the levels of TNF-α, IL-1β, andCXCL10; or the method comprises reducing the levels of IL-6, IL-1β, andCXCL10; or the method comprises reducing the levels of IL-6, IL-1β,TNF-α, and CXCL10.

In some embodiments, methods of treating conditions associated with anelevated level of at least one, at least two, at least three, or atleast four, at least five, at least six, at least seven, at least eight,at least nine, or at least ten factors selected from IL-6, IL-1β, IL-8,CCL2, CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9are provided. In some embodiments, a method comprises administering aneffective amount of an antibody that binds colony stimulating factor 1receptor (CSF1R) to a subject with the condition, wherein the antibodyblocks binding of colony stimulating factor 1 (CSF1) to CSF1R and blocksbinding of IL-34 to CSF1R. In some embodiments, the antibody reduces thelevel of at least one, at least two, at least three, or at least four,at least five, at least six, at least seven, at least eight, at leastnine, or at least ten factors selected from IL-6, IL-1β, IL-8, CCL2,CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9. Insome embodiments, the subject has a condition selected from rheumatoidarthritis, juvenile idiopathic arthritis, Castleman's disease,psoriasis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease,and ulcerative colitis, lupus erythematosus, and inflammatory boweldisease. In some embodiments, a condition is associated with an elevatedlevel of at least one, at least two, at least three, or four factorsselected from IL-6, IL-1β, TNF-α, and CXCL10. In some embodiments, acondition is associated with an elevated level of IL-6. In some suchembodiments, the condition is selected from rheumatoid arthritis,juvenile idiopathic arthritis, and Castleman's disease. In someembodiments, a condition is associated with an elevated level of TNF-α.In some such embodiments, the condition is selected from rheumatoidarthritis, juvenile idiopathic arthritis, psoriasis, psoriaticarthritis, ankylosing spondylitis, Crohn's disease, and ulcerativecolitis. In some embodiments, a condition is associated with an elevatedlevel of IL-1β. In some such embodiments, the condition is selected fromrheumatoid arthritis and juvenile idiopathic arthritis. In someembodiments, a condition is associated with an elevated level of CXCL10.

In some embodiments, a method of treating inflammatory arthritis isprovided. In some embodiments, the method comprises administering aneffective amount of an antibody that binds CSF1R to a subject withinflammatory arthritis, wherein the antibody blocks binding of CSF1 toCSF1R and blocks binding of IL-34 to CSF1R, and wherein the antibodyreduces the level of at least one, at least two, at least three, or atleast four, at least five, at least six, at least seven, at least eight,at least nine, or at least ten factors selected from IL-6, IL-1β, IL-8,CCL2, CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9.In some embodiments, the inflammatory arthritis is selected fromrheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, andjuvenile idiopathic arthritis.

In some embodiments, methods of treating an inflammatory condition areprovided. In some embodiments, a method comprises administering aneffective amount of an antibody that binds CSF1R to a subject with aninflammatory condition, wherein the antibody blocks binding of CSF1 toCSF1R and blocks binding of IL-34 to CSF1R, and wherein the antibodyreduces the level of at least one, at least two, at least three, or atleast four, at least five, at least six, at least seven, at least eight,at least nine, or at least ten factors selected from IL-6, IL-1β, IL-8,CCL2, CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9.In some embodiments, the inflammatory condition is selected fromrheumatoid arthritis, juvenile idiopathic arthritis, Castleman'sdisease, psoriasis, psoriatic arthritis, ankylosing spondylitis, Crohn'sdisease, and ulcerative colitis, lupus erythematosus, and inflammatorybowel disease.

In some embodiments, methods of treating CD16+ disorder are provided. Insome embodiments, a method comprises administering an effective amountof an antibody that binds CSF1R to a subject with a CD16+ disorder,wherein the antibody blocks binding of CSF1 to CSF1R and blocks bindingof IL-34 to CSF1R, and wherein the antibody reduces the level of atleast one, at least two, at least three, or at least four, at leastfive, at least six, at least seven, at least eight, at least nine, or atleast ten factors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α,CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9. In some embodiments,the antibody reduces the level of at least one, at least two, at leastthree, or four factors selected from IL-6, IL-1β, TNF-α, and CXCL10. Insome embodiments, the CD16+ disorder is selected from rheumatoidarthritis, juvenile idiopathic arthritis, Castleman's disease,psoriasis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease,and ulcerative colitis, lupus erythematosus, and inflammatory boweldisease. In some embodiments, the antibody substantially reduces thenumber of CD16+ monocytes. In some embodiments, the number of CD16−monocytes are substantially unchanged following administration of theantibody.

In any of the embodiments described herein, the antibody may reduce thelevel of at least one, at least two, at least three, or at least four,at least five, at least six, at least seven, at least eight, at leastnine, or at least ten factors selected from IL-6, IL-1β, IL-8, CCL2,CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9 invitro.

In any of the embodiments described herein, the subject may have anelevated level of at least one, at least two, at least three, or atleast four, at least five, at least six, at least seven, at least eight,at least nine, or at least ten factors selected from IL-6, IL-1β, IL-8,CCL2, CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9prior to administration of the antibody.

In some embodiments, a method further comprises administering at leastone additional therapeutic agent selected from methotrexate, an anti-TNFagent, a glucocorticoid, cyclosporine, leflunomide, azathioprine, a JAKinhibitor, a SYK inhibitor, an anti-IL-6 agent, an anti-CD20 agent, ananti-CD19 agent, an anti-GM-CSF agent, an anti-IL-1 agent, and a CTLA4agent. In some embodiments, the at least one additional therapeuticagent is selected from methotrexate, an anti-TNF-α antibody, a solubleTNF receptor, a glucocorticoid, cyclosporine, leflunomide, azathioprine,a JAK inhibitor, a SYK inhibitor, an anti-IL-6 antibody, an anti-IL-6receptor antibody, an anti-CD20 antibody, an anti-CD19 antibody, ananti-GM-CSF antibody, and anti-GM-CSF receptor antibody, an anti-IL-1antibody, an IL-1 receptor antagonist, and a CTLA4-Ig fusion molecule.In some embodiments, the condition is resistant to methotrexate.

In some embodiments, a method of treating an inflammatory condition isprovided, wherein the method comprises (a) determining the level of atleast one, at least two, at least three, or at least four, at leastfive, at least six, at least seven, at least eight, at least nine, or atleast ten factors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α,CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9 in a subject with theinflammatory condition; and (b) if the level of at least one of thefactors is elevated in the subject, administering to the subject aneffective amount of an antibody that binds CSF1R and blocks binding ofIL-34 to CSF1R, wherein the antibody reduces the level of at least one,at least two, at least three, or at least four, at least five, at leastsix, at least seven, at least eight, at least nine, or at least tenfactors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7,CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9.

In some embodiments, a method of treating an inflammatory condition isprovided, wherein the method comprises (a) detecting an elevated levelof at least one, at least two, at least three, or at least four, atleast five, at least six, at least seven, at least eight, at least nine,or at least ten factors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10,TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9 in a subjectwith the inflammatory condition; and (b) administering to the subject aneffective amount of an antibody that binds CSF1R and blocks binding ofIL-34 to CSF1R, wherein the antibody reduces the level of at least one,at least two, at least three, or at least four, at least five, at leastsix, at least seven, at least eight, at least nine, or at least tenfactors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7,CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9.

In any of the methods described herein, the antibody may reduce thelevel of IL-6; or the antibody may reduce the level of TNF-α; or theantibody may reduce the level of IL-1β; or the antibody may reduce thelevel of CXCL10; or the antibody may reduce the levels of IL-6 andTNF-α; or the antibody may reduce the levels of IL-6 and IL-1β; or theantibody may reduce the levels of IL-6 and CXCL10; or the antibody mayreduce the levels of TNF-α and IL-1β; or the antibody may reduce thelevels of TNF-α and CXCL10; or the antibody may reduce the levels ofIL-1β and CXCL10; or the antibody may reduce the levels of IL-6, TNF-α,and IL-1β; or the antibody may reduce the levels of IL-6, TNF-α, andCXCL10; or the antibody may reduce the levels of TNF-α, IL-1β, andCXCL10; or the method comprises reducing the levels of IL-6, IL-1β, andCXCL10; or the antibody may reduce the levels of IL-6, IL-1β, TNF-α, andCXCL10.

In some embodiments, a method of identifying a subject who may benefitfrom an antibody that binds CSF1R, wherein the antibody blocks bindingof CSF1 to CSF1R and blocks binding of IL-34 to CSF1R is provided,comprising determining the level of at least one, at least two, at leastthree, or at least four, at least five, at least six, at least seven, atleast eight, at least nine, or at least ten factors selected from IL-6,IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7,MMP-2, and MMP-9 in the subject, wherein an elevated level of at leastone of the factors in the subject indicates that the subject may benefitfrom the antibody that binds CSF1R. In some embodiments, the subject hasa CD16+ disorder. In some embodiments, the subject has rheumatoidarthritis. In some embodiments, the subject has an elevated level ofCD16+ monocytes.

In some embodiments, a method of predicting responsiveness in a subjectsuffering from an inflammatory condition to an antibody that bindsCSF1R, wherein the antibody blocks binding of CSF1 to CSF1R and blocksbinding of IL-34 to CSF1R is provided, comprising determining the levelof at least one, at least two, at least three, or at least four, atleast five, at least six, at least seven, at least eight, at least nine,or at least ten factors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10,TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9 in thesubject, wherein an elevated level of at least one of the factors in thesubject indicates that the subject is more likely to respond to theantibody that binds CSF1R. In some embodiments, the subject has a CD16+disorder. In some embodiments, the subject has rheumatoid arthritis. Insome embodiments, the subject has an elevated level of CD16+ monocytes.

In any of the embodiments described herein, a condition may be resistantto methotrexate and/or the subject may be a methotrexate inadequateresponder. Further, in any of the embodiments described herein, acondition may be resistant to a TNF inhibitor and/or the subject may bea TNF inhibitor inadequate responder.

In some embodiments, a method of treating a methotrexate inadequateresponder is provided, comprising administering to the methotrexateinadequate responder an antibody that binds CSF1R, wherein the antibodyblocks binding of CSF1 to CSF1R and blocks binding of IL-34 to CSF1R. Insome embodiments, a method of treating a TNF inhibitor inadequateresponder is provided, comprising administering to the TNF inhibitorinadequate responder an antibody that binds CSF1R, wherein the antibodyblocks binding of CSF1 to CSF1R and blocks binding of IL-34 to CSF1R. Insome embodiments, the inadequate responder has a CD16+ disorder. In someembodiments, the CD16+ disorder is rheumatoid arthritis. In someembodiments, the antibody substantially reduces the number of CD16+monocytes. In some embodiments, the number of CD16− monocytes aresubstantially unchanged following administration of the antibody. Insome embodiments, the level of at least one, at least two, at leastthree, or at least four, at least five, at least six, at least seven, atleast eight, at least nine, or at least ten factors selected from IL-6,IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7,MMP-2, and MMP-9 in the methotrexate and/or TNF-inhibitor inadequateresponder is reduced following administration of the antibody.

In any of the methods described herein, the antibody heavy chain and/orthe antibody light chain may have the structure described below.

In any of the methods described herein, the antibody heavy chain maycomprise a sequence that is at least 90%, at least 95%, at least 97%, atleast 99%, or 100% identical to a sequence selected from SEQ ID NOs: 9,11, 13, and 39 to 45. In any of the methods described herein, theantibody light chain may comprise a sequence that is at least 90%, atleast 95%, at least 97%, at least 99%, or 100% identical to a sequenceselected from SEQ ID NOs: 10, 12, 14, and 46 to 52. In any of themethods described herein, the antibody heavy chain may comprise asequence that is at least 90%, at least 95%, at least 97%, at least 99%,or 100% identical to a sequence selected from SEQ ID NOs: 9, 11, 13, and39 to 45, and the antibody light chain may comprise a sequence that isat least 90%, at least 95%, at least 97%, at least 99%, or 100%identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to52.

In any of the methods described herein, the HC CDR1, HC CDR2, and HCCDR3 may comprise a set of sequences selected from: (a) SEQ ID NOs: 15,16, and 17; (b) SEQ ID NOs: 21, 22, and 23; and (c) SEQ ID NOs: 27, 28,and 29. In any of the methods described herein, the LC CDR1, LC CDR2,and LC CDR3 may comprise a set of sequences selected from: (a) SEQ IDNOs: 18, 19, and 20; (b) SEQ ID NOs: 24, 25, and 26; and (c) SEQ ID NOs:30, 31, and 32.

In any of the methods described herein, the heavy chain may comprise anHC CDR1, HC CDR2, and HC CDR3, wherein the HC CDR1, HC CDR2, and HC CDR3comprise a set of sequences selected from: (a) SEQ ID NOs: 15, 16, and17; (b) SEQ ID NOs: 21, 22, and 23; and (c) SEQ ID NOs: 27, 28, and 29;and the light chain may comprise an LC CDR1, LC CDR2, and LC CDR3,wherein the LC CDR1, LC CDR2, and LC CDR3 comprise a set of sequencesselected from: (a) SEQ ID NOs: 18, 19, and 20; (b) SEQ ID NOs: 24, 25,and 26; and (c) SEQ ID NOs: 30, 31, and 32.

In any of the methods described herein, the antibody that binds CSF1Rmay comprise: (a) a heavy chain comprising a sequence that is at least95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 9 and alight chain comprising a sequence that is at least 95%, at least 97%, atleast 99%, or 100% identical to SEQ ID NO: 10; (b) a heavy chaincomprising a sequence that is at least 95%, at least 97%, at least 99%,or 100% identical to SEQ ID NO: 11 and a light chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 12; (c) a heavy chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 13 and a light chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 14; (d) a heavychain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 39 and a light chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 46; (e) a heavy chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 40 and a light chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 46; (f) a heavychain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 41 and a light chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 46; (g) a heavy chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 39 and a light chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 47; (h) a heavychain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 40 and a light chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 47; (i) a heavy chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 41 and a light chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 47; and (j) aheavy chain comprising a sequence that is at least 95%, at least 97%, atleast 99%, or 100% identical to SEQ ID NO: 42 and a light chaincomprising a sequence that is at least 95%, at least 97%, at least 99%,or 100% identical to SEQ ID NO: 48; (k) a heavy chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 42 and a light chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 49; (l) a heavy chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 42 and a lightchain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 50; (m) a heavy chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 43 and a light chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 48; (n) a heavy chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 43 and a lightchain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 49; (o) a heavy chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 43 and a light chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 50; (p) a heavy chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 44 and a lightchain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 51; (q) a heavy chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 44 and a light chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 52; (r) a heavy chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 45 and a lightchain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 51; or (s) a heavy chain comprisinga sequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 45 and a light chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 52.

In any of the methods described herein, the antibody may comprise: (a) aheavy chain comprising a heavy chain (HC) CDR1 having the sequence ofSEQ ID NO: 15, an HC CDR2 having the sequence of SEQ ID NO: 16, and anHC CDR3 having the sequence of SEQ ID NO: 17, and a light chaincomprising a light chain (LC) CDR1 having the sequence of SEQ ID NO: 18,a LC CDR2 having the sequence of SEQ ID NO: 19, and a LC CDR3 having thesequence of SEQ ID NO: 20; (b) a heavy chain comprising a heavy chain(HC) CDR1 having the sequence of SEQ ID NO: 21, an HC CDR2 having thesequence of SEQ ID NO: 22, and an HC CDR3 having the sequence of SEQ IDNO: 23, and a light chain comprising a light chain (LC) CDR1 having thesequence of SEQ ID NO: 24, a LC CDR2 having the sequence of SEQ ID NO:25, and a LC CDR3 having the sequence of SEQ ID NO: 26; or (c) a heavychain comprising a heavy chain (HC) CDR1 having the sequence of SEQ IDNO: 27, an HC CDR2 having the sequence of SEQ ID NO: 28, and an HC CDR3having the sequence of SEQ ID NO: 29, and a light chain comprising alight chain (LC) CDR1 having the sequence of SEQ ID NO: 30, a LC CDR2having the sequence of SEQ ID NO: 31, and a LC CDR3 having the sequenceof SEQ ID NO: 32.

In any of the methods described herein, the antibody may comprise: (a) aheavy chain comprising a sequence of SEQ ID NO: 53 and a light chaincomprising a sequence of SEQ ID NO: 60; (b) a heavy chain comprising asequence of SEQ ID NO: 53 and a light chain comprising a sequence of SEQID NO: 61; or (c) a heavy chain comprising a sequence of SEQ ID NO: 58and a light chain comprising a sequence of SEQ ID NO: 65. In someembodiments, an antibody comprises a heavy chain and a light chain,wherein the antibody comprises: (a) a heavy chain consisting of thesequence of SEQ ID NO: 53 and a light chain consisting of the sequenceof SEQ ID NO: 60; (b) a heavy chain consisting of the sequence of SEQ IDNO: 53 and a light chain consisting of the sequence of SEQ ID NO: 61; or(c) a heavy chain consisting of the sequence of SEQ ID NO: 58 and alight chain consisting of the sequence of SEQ ID NO: 65.

In any of the methods described herein, the antibody may be a humanizedantibody. In any of the methods described herein, the antibody may beselected from a Fab, an Fv, an scFv, a Fab′, and a (Fab′)₂. In any ofthe methods described herein, the antibody may be a chimeric antibody.In any of the methods described herein, the antibody may be selectedfrom an IgA, an IgG, and an IgD. In any of the methods described herein,the antibody may be an IgG. In any of the methods described herein, theantibody may be an IgG4. In any of the methods described herein, theantibody may be an IgG4 comprising an S241P mutation in at least oneIgG4 heavy chain constant region.

In any of the methods described herein, the antibody may bind to humanCSF1R and/or binds to cynomolgus CSF1R. In any of the methods describedherein, the antibody may block ligand binding to CSF1R. In any of themethods described herein, the antibody may block binding of CSF1 and/orIL-34 to CSF1R. In any of the methods described herein, the antibody mayblock binding of both CSF1 and IL-34 to CSF1R. In any of the methodsdescribed herein, the antibody may inhibit ligand-induced CSF1Rphosphorylation. In any of the methods described herein, the antibodymay inhibit CSF1- and/or IL-34-induced CSF1R phosphorylation. In any ofthe methods described herein, the antibody may bind to human CSF1R withan affinity (K_(D)) of less than 1 nM. In any of the methods describedherein, the antibody may inhibit monocyte proliferation and/or survivalresponses in the presence of CSF1 or IL-34.

In some embodiments, a pharmaceutical composition comprising an antibodythat binds CSF1R is provided. In some embodiments, antibodies that bindCSF1R and compositions comprising antibodies that bind CSF1R areprovided for use in any of the methods of treatment described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-C show an alignment of the humanized heavy chain variableregions for each of humanized antibodies huAb1 to huAb16, as discussedin Example 1, with FIG. 1A showing alignment of positions 1-43, FIG. 1Bshowing alignment of positions 44-82, and FIG. 1C showing alignment ofpositions 82a-113. Boxed residues are amino acids in the human acceptorsequence that were changed back to the corresponding mouse residue.

FIG. 2A-C show an alignment of the humanized light chain variableregions for each of humanized antibodies huAb1 to huAb16, as discussedin Example 1, with FIG. 2A showing alignment of positions 1-34, FIG. 2Bshowing alignment of positions 35-70, and FIG. 2C showing alignment ofpositions 71-107. Boxed amino acids are residues in the human acceptorsequence that were changed back to the corresponding mouse residue.

FIG. 3 shows IL-6 cytokine concentration determined by ELISA on tissueculture media of intact synovial explants (n=6 patients with rheumatoidarthritis) treated for 4 days with 1 μg/ml huAb1 or IgG4 isotypecontrol, as described in Example 2.

FIG. 4A-L show cytokine and matrix metalloproteinase concentrationsdetermined by multiplex Luminex® analysis on tissue culture media ofintact synovial explants (n=4 patients with rheumatoid arthritis)treated for 4 days with huAb1 or IgG4 isotype control, as described inExample 2. FIG. 4A shows IL-6, FIG. 4B shows IL-1beta, FIG. 4C showsIL-8, FIG. 4D shows CCL2/MCP-1, FIG. 4E shows CXCL-10; FIG. 4F showsTNF-alpha, FIG. 4G shows MCP-3; FIG. 4H shows CXCL-5, FIG. 4I showsCXCL-9; FIG. 4J shows CXCL-6, FIG. 4K shows MMP-7; and FIG. 4L showsMMP-9.

DETAILED DESCRIPTION

Methods of reducing the level of one or more factors selected from IL-6,IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7,MMP-2, and MMP-9 in a subject comprising administering antibodies thatbind CSF1R and block CSF1 and IL-34 ligand binding are provided. Asdiscussed herein, antibodies that bind CSF1R and block CSF1 and IL-34ligand binding are effective for reducing the levels of one or morefactors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7,CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9 and treating conditionsassociated with elevated levels of those factors. Exemplary suchconditions include, but are not limited to, rheumatoid arthritis,juvenile idiopathic arthritis, Castleman's disease, psoriasis, psoriaticarthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis,lupus erythematosus, and inflammatory bowel disease. The presentinventors found that contacting synovial biopsy samples from rheumatoidarthritis patients with an antibody that binds CSF1R reduces the levelsof IL-6, IL-1β, IL-8, CCL2 (also referred to as MCP-1), CXCL10, TNF-α,CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All references cited herein, including patent applications andpublications, are incorporated herein by reference in their entiretiesfor any purpose.

Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Exemplary techniques used in connection with recombinant DNA,oligonucleotide synthesis, tissue culture and transformation (e.g.,electroporation, lipofection), enzymatic reactions, and purificationtechniques are known in the art. Many such techniques and procedures aredescribed, e.g., in Sambrook et al. Molecular Cloning: A LaboratoryManual (2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989)), among other places. In addition, exemplarytechniques for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients arealso known in the art.

In this application, the use of “or” means “and/or” unless statedotherwise. In the context of a multiple dependent claim, the use of “or”refers back to more than one preceding independent or dependent claim inthe alternative only. Also, terms such as “element” or “component”encompass both elements and components comprising one unit and elementsand components that comprise more than one subunit unless specificallystated otherwise.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The terms “nucleic acid molecule” and “polynucleotide” may be usedinterchangeably, and refer to a polymer of nucleotides. Such polymers ofnucleotides may contain natural and/or non-natural nucleotides, andinclude, but are not limited to, DNA, RNA, and PNA. “Nucleic acidsequence” refers to the linear sequence of nucleotides that comprise thenucleic acid molecule or polynucleotide.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Such polymers of amino acid residues may contain natural ornon-natural amino acid residues, and include, but are not limited to,peptides, oligopeptides, dimers, trimers, and multimers of amino acidresidues. Both full-length proteins and fragments thereof areencompassed by the definition. The terms also include post-expressionmodifications of the polypeptide, for example, glycosylation,sialylation, acetylation, phosphorylation, and the like. Furthermore,for purposes of the present invention, a “polypeptide” refers to aprotein which includes modifications, such as deletions, additions, andsubstitutions (generally conservative in nature), to the nativesequence, as long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe proteins or errors due to PCR amplification.

The term “CSF1R” refers herein to the full-length CSF1R, which includesthe N-terminal ECD, the transmembrane domain, and the intracellulartyrosine kinase domain, with or without an N-terminal leader sequence.In some embodiments, the CSF1R is a human CSF1R having the amino acidsequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The term “CSF1R extracellular domain” (“CSF1R ECD”) as used hereinrefers to a CSF1R polypeptide that lacks the intracellular andtransmembrane domains. CSF1R ECDs include the full-length CSF1R ECD andCSF1R ECD fragments that are capable of binding CSF1 and/or IL-34. Thehuman full-length CSF1R ECD is defined herein as comprising either aminoacids 1 to 512 (i.e., including the leader sequence) or amino acids 20to 512 (i.e., lacking the leader sequence) of SEQ ID NO: 2. In someembodiments, a human CSF1R ECD fragment comprises amino acids 20 to 506of SEQ ID NO: 2 (see SEQ ID NO: 5). In some embodiments, a human CSF1Rfragment ends at amino acid 507, 508, 509, 510, or 511. In someembodiments, a cyno CSF1R ECD comprises the sequence of SEQ ID NO: 7(with leader sequence) or amino acids 20 to 506 of SEQ ID NO: 7 (withoutleader sequence).

With reference to anti-CSF1R antibodies the term “blocks binding of” aligand, such as CSF1 and/or IL-34, and grammatical variants thereof, areused to refer to the ability to inhibit the interaction between CSF1Rand a CSF1R ligand, such as CSF1 and/or IL-34. Such inhibition may occurthrough any mechanism, including direct interference with ligandbinding, e.g., because of overlapping binding sites on CSF1R, and/orconformational changes in CSF1R induced by the antibody that alterligand affinity, etc. Antibodies and antibody fragments referred to as“functional” are characterized by having such properties.

An “immunological” activity refers only to the ability to induce theproduction of an antibody against an antigenic epitope possessed by anative or naturally-occurring CSF1R polypeptide.

The term “antibody” as used herein refers to a molecule comprising atleast complementarity-determining region (CDR) 1, CDR2, and CDR3 of aheavy chain and at least CDR1, CDR2, and CDR3 of a light chain, whereinthe molecule is capable of binding to antigen. The term antibodyincludes, but is not limited to, fragments that are capable of bindingantigen, such as Fv, single-chain Fv (scFv), Fab, Fab′, and (Fab′)₂. Theterm antibody also includes, but is not limited to, chimeric antibodies,humanized antibodies, and antibodies of various species such as mouse,human, cynomolgus monkey, etc.

In some embodiments, an antibody comprises a heavy chain variable regionand a light chain variable region. In some embodiments, an antibodycomprises at least one heavy chain comprising a heavy chain variableregion and at least a portion of a heavy chain constant region, and atleast one light chain comprising a light chain variable region and atleast a portion of a light chain constant region. In some embodiments,an antibody comprises two heavy chains, wherein each heavy chaincomprises a heavy chain variable region and at least a portion of aheavy chain constant region, and two light chains, wherein each lightchain comprises a light chain variable region and at least a portion ofa light chain constant region. As used herein, a single-chain Fv (scFv),or any other antibody that comprises, for example, a single polypeptidechain comprising all six CDRs (three heavy chain CDRs and three lightchain CDRs) is considered to have a heavy chain and a light chain. Insome such embodiments, the heavy chain is the region of the antibodythat comprises the three heavy chain CDRs and the light chain in theregion of the antibody that comprises the three light chain CDRs.

The term “heavy chain variable region” as used herein refers to a regioncomprising heavy chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. Insome embodiments, a heavy chain variable region also comprises at leasta portion of an FR1 and/or at least a portion of an FR4. In someembodiments, a heavy chain CDR1 corresponds to Kabat residues 26 to 35;a heavy chain CDR2 corresponds to Kabat residues 50 to 65; and a heavychain CDR3 corresponds to Kabat residues 95 to 102. See, e.g., KabatSequences of Proteins of Immunological Interest (1987 and 1991, NIH,Bethesda, Md.); and FIGS. 1A-C. In some embodiments, a heavy chain CDR1corresponds to Kabat residues 31 to 35; a heavy chain CDR2 correspondsto Kabat residues 50 to 65; and a heavy chain CDR3 corresponds to Kabatresidues 95 to 102. See id.

The term “heavy chain constant region” as used herein refers to a regioncomprising at least three heavy chain constant domains, C_(H)1, C_(H)2,and C_(H)3. Nonlimiting exemplary heavy chain constant regions includeγ, δ, and α. Nonlimiting exemplary heavy chain constant regions alsoinclude ε and μ. Each heavy constant region corresponds to an antibodyisotype. For example, an antibody comprising a γ constant region is anIgG antibody, an antibody comprising a δ constant region is an IgDantibody, and an antibody comprising an α constant region is an IgAantibody. Further, an antibody comprising a μ constant region is an IgMantibody, and an antibody comprising an ε constant region is an IgEantibody. Certain isotypes can be further subdivided into subclasses.For example, IgG antibodies include, but are not limited to, IgG1(comprising a γ₁ constant region), IgG2 (comprising a γ₂ constantregion), IgG3 (comprising a γ₃ constant region), and IgG4 (comprising aγ₄ constant region) antibodies; IgA antibodies include, but are notlimited to, IgA1 (comprising an α₁ constant region) and IgA2 (comprisingan α₂ constant region) antibodies; and IgM antibodies include, but arenot limited to, IgM1 and IgM2.

In some embodiments, a heavy chain constant region comprises one or moremutations (or substitutions), additions, or deletions that confer adesired characteristic on the antibody. A nonlimiting exemplary mutationis the S241P mutation in the IgG4 hinge region (between constant domainsC_(H)1 and C_(H)2), which alters the IgG4 motif CPSCP to CPPCP, which issimilar to the corresponding motif in IgG1. That mutation, in someembodiments, results in a more stable IgG4 antibody. See, e.g., Angal etal., Mol. Immunol. 30: 105-108 (1993); Bloom et al., Prot. Sci. 6:407-415 (1997); Schuurman et al., Mol. Immunol. 38: 1-8 (2001).

The term “heavy chain” as used herein refers to a polypeptide comprisingat least a heavy chain variable region, with or without a leadersequence. In some embodiments, a heavy chain comprises at least aportion of a heavy chain constant region. The term “full-length heavychain” as used herein refers to a polypeptide comprising a heavy chainvariable region and a heavy chain constant region, with or without aleader sequence.

The term “light chain variable region” as used herein refers to a regioncomprising light chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. Insome embodiments, a light chain variable region also comprises an FR1and/or an FR4. In some embodiments, a light chain CDR1 corresponds toKabat residues 24 to 34; a light chain CDR2 corresponds to Kabatresidues 50 to 56; and a light chain CDR3 corresponds to Kabat residues89 to 97. See, e.g., Kabat Sequences of Proteins of ImmunologicalInterest (1987 and 1991, NIH, Bethesda, Md.); and FIGS. 1A-C.

The term “light chain constant region” as used herein refers to a regioncomprising a light chain constant domain, C_(L). Nonlimiting exemplarylight chain constant regions include λ and κ.

The term “light chain” as used herein refers to a polypeptide comprisingat least a light chain variable region, with or without a leadersequence. In some embodiments, a light chain comprises at least aportion of a light chain constant region. The term “full-length lightchain” as used herein refers to a polypeptide comprising a light chainvariable region and a light chain constant region, with or without aleader sequence.

A “chimeric antibody” as used herein refers to an antibody comprising atleast one variable region from a first species (such as mouse, rat,cynomolgus monkey, etc.) and at least one constant region from a secondspecies (such as human, cynomolgus monkey, etc.). In some embodiments, achimeric antibody comprises at least one mouse variable region and atleast one human constant region. In some embodiments, a chimericantibody comprises at least one cynomolgus variable region and at leastone human constant region. In some embodiments, a chimeric antibodycomprises at least one rat variable region and at least one mouseconstant region. In some embodiments, all of the variable regions of achimeric antibody are from a first species and all of the constantregions of the chimeric antibody are from a second species.

A “humanized antibody” as used herein refers to an antibody in which atleast one amino acid in a framework region of a non-human variableregion has been replaced with the corresponding amino acid from a humanvariable region. In some embodiments, a humanized antibody comprises atleast one human constant region or fragment thereof. In someembodiments, a humanized antibody is a Fab, an scFv, a (Fab′)₂, etc.

A “CDR-grafted antibody” as used herein refers to a humanized antibodyin which the complementarity determining regions (CDRs) of a first(non-human) species have been grafted onto the framework regions (FRs)of a second (human) species.

A “human antibody” as used herein refers to antibodies produced inhumans, antibodies produced in non-human animals that comprise humanimmunoglobulin genes, such as XenoMouse®, and antibodies selected usingin vitro methods, such as phage display, wherein the antibody repertoireis based on a human immunoglobulin sequences.

The term “leader sequence” refers to a sequence of amino acid residueslocated at the N terminus of a polypeptide that facilitates secretion ofa polypeptide from a mammalian cell. A leader sequence may be cleavedupon export of the polypeptide from the mammalian cell, forming a matureprotein. Leader sequences may be natural or synthetic, and they may beheterologous or homologous to the protein to which they are attached.Exemplary leader sequences include, but are not limited to, antibodyleader sequences, such as, for example, the amino acid sequences of SEQID NOs: 3 and 4, which correspond to human light and heavy chain leadersequences, respectively. Nonlimiting exemplary leader sequences alsoinclude leader sequences from heterologous proteins. In someembodiments, an antibody lacks a leader sequence. In some embodiments,an antibody comprises at least one leader sequence, which may beselected from native antibody leader sequences and heterologous leadersequences.

The term “vector” is used to describe a polynucleotide that may beengineered to contain a cloned polynucleotide or polynucleotides thatmay be propagated in a host cell. A vector may include one or more ofthe following elements: an origin of replication, one or more regulatorysequences (such as, for example, promoters and/or enhancers) thatregulate the expression of the polypeptide of interest, and/or one ormore selectable marker genes (such as, for example, antibioticresistance genes and genes that may be used in colorimetric assays,e.g., β-galactosidase). The term “expression vector” refers to a vectorthat is used to express a polypeptide of interest in a host cell.

A “host cell” refers to a cell that may be or has been a recipient of avector or isolated polynucleotide. Host cells may be prokaryotic cellsor eukaryotic cells. Exemplary eukaryotic cells include mammalian cells,such as primate or non-primate animal cells; fungal cells, such asyeast; plant cells; and insect cells. Nonlimiting exemplary mammaliancells include, but are not limited to, NSO cells, PER.C6® cells(Crucell), and 293 and CHO cells, and their derivatives, such as 293-6Eand DG44 cells, respectively.

The term “isolated” as used herein refers to a molecule that has beenseparated from at least some of the components with which it istypically found in nature. For example, a polypeptide is referred to as“isolated” when it is separated from at least some of the components ofthe cell in which it was produced. Where a polypeptide is secreted by acell after expression, physically separating the supernatant containingthe polypeptide from the cell that produced it is considered to be“isolating” the polypeptide. Similarly, a polynucleotide is referred toas “isolated” when it is not part of the larger polynucleotide (such as,for example, genomic DNA or mitochondrial DNA, in the case of a DNApolynucleotide) in which it is typically found in nature, or isseparated from at least some of the components of the cell in which itwas produced, e.g., in the case of an RNA polynucleotide. Thus, a DNApolynucleotide that is contained in a vector inside a host cell may bereferred to as “isolated” so long as that polynucleotide is not found inthat vector in nature.

The term “elevated level” means a higher level of a protein, such as acytokine or matrix metalloproteinase, in a particular tissue of asubject relative to the same tissue in a control, such as an individualor individuals who are not suffering from an inflammatory condition orother condition described herein. The elevated level may be the resultof any mechanism, such as increased expression, increased stability,decreased degradation, increased secretion, decreased clearance, etc.,of the protein.

The term “reduce” or “reduces” means to lower the level of a protein,such as a cytokine or matrix metalloproteinase, in a particular tissueof a subject by at least 10%. In some embodiments, an agent, such as anantibody that binds CSF1R, reduces the level of a protein, such as acytokine or matrix metalloproteinase, in a particular tissue of asubject by at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, or at least 90%. In some embodiments, the level of a proteinis reduced relative to the level of the protein prior to contacting withan agent, such as an antibody that binds CSF1R.

The term “resistant,” when used in the context of resistance to atherapeutic agent, means a decreased response or lack of response to astandard dose of the therapeutic agent, relative to the subject'sresponse to the standard dose of the therapeutic agent in the past, orrelative to the expected response of a similar subject with a similardisorder to the standard dose of the therapeutic agent. Thus, in someembodiments, a subject may be resistant to therapeutic agent althoughthe subject has not previously been given the therapeutic agent, or thesubject may develop resistance to the therapeutic agent after havingresponded to the agent on one or more previous occasions.

The terms “subject” and “patient” are used interchangeably herein torefer to a human. In some embodiments, methods of treating othermammals, including, but not limited to, rodents, simians, felines,canines, equines, bovines, porcines, ovines, caprines, mammalianlaboratory animals, mammalian farm animals, mammalian sport animals, andmammalian pets, are also provided.

The term “sample,” as used herein, refers to a composition that isobtained or derived from a subject that contains a cellular and/or othermolecular entity that is to be characterized, quantitated, and/oridentified, for example based on physical, biochemical, chemical and/orphysiological characteristics. An exemplary sample is a tissue sample.

The term “tissue sample” refers to a collection of similar cellsobtained from a tissue of a subject. The source of the tissue sample maybe solid tissue as from a fresh, frozen and/or preserved organ or tissuesample or biopsy or aspirate; blood or any blood constituents; bodilyfluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid,synovial fluid, or interstitial fluid; cells from any time in gestationor development of the subject. In some embodiments, a tissue sample is asynovial biopsy tissue sample and/or a synovial fluid sample. In someembodiments, a tissue sample is a synovial fluid sample. The tissuesample may also be primary or cultured cells or cell lines. Optionally,the tissue sample is obtained from a disease tissue/organ. The tissuesample may contain compounds that are not naturally intermixed with thetissue in nature such as preservatives, anticoagulants, buffers,fixatives, nutrients, antibiotics, or the like. A “control sample” or“control tissue”, as used herein, refers to a sample, cell, or tissueobtained from a source known, or believed, not to be afflicted with thedisease for which the subject is being treated.

For the purposes herein a “section” of a tissue sample means a part orpiece of a tissue sample, such as a thin slice of tissue or cells cutfrom a solid tissue sample.

As used herein, “rheumatoid arthritis” or “RA” refers to a recognizeddisease state that may be diagnosed according to the 1987, 2000, or 2010criteria for the classification of RA (American Rheumatism Associationor Americal College of Rheumatology), or any similar criteria. In someembodiments, the term “rheumatoid arthritis” refers to a chronicautoimmune disease characterized primarily by inflammation of the lining(synovium) of the joints, which can lead to joint damage, resulting inchronic pain, loss of function, and disability. Because RA can affectmultiple organs of the body, including skin, lungs, and eyes, it isreferred to as a systemic illness.

The term “rheumatoid arthritis” includes not only active and early RA,but also incipient RA, as defined below. Physiological indicators of RAinclude, symmetric joint swelling which is characteristic though notinvariable in RA. Fusiform swelling of the proximal interphalangeal(PIP) joints of the hands as well as metacarpophalangeal (MCP), wrists,elbows, knees, ankles, and metatarsophalangeal (MTP) joints are commonlyaffected and swelling is easily detected. Pain on passive motion is themost sensitive test for joint inflammation, and inflammation andstructural deformity often limits the range of motion for the affectedjoint. Typical visible changes include ulnar deviation of the fingers atthe MCP joints, hyperextension, or hyperflexion of the MCP and PIPjoints, flexion contractures of the elbows, and subluxation of thecarpal bones and toes. The subject with RA may be resistant to adisease-modifying anti-rheumatic drug (DMARD), and/or a non-steroidalanti-inflammatory drug (NSAID). Nonlimiting exemplary “DMARDs” includehydroxychloroquine, sulfasalazine, methotrexate (MTX), leflunomide,etanercept, infliximab (plus oral and subcutaneous MTX), azathioprine,D-penicillamine, gold salts (oral), gold salts (intramuscular),minocycline, cyclosporine including cyclosporine A and topicalcyclosporine, staphylococcal protein A (Goodyear and Silverman, J. Exp.Med., 197(9):1125-1139 (2003)), including salts and derivatives thereof,etc. Further candidates for therapy according to this invention includethose who have experienced an inadequate response to previous or currenttreatment with TNF inhibitors such as etanercept, infliximab, golimumab,certolizumab, and/or adalimumab because of toxicity, inadequateefficacy, and/or resistance.

A patient with “active rheumatoid arthritis” means a patient with activeand not latent symptoms of RA. Subjects with “early active rheumatoidarthritis” are those subjects with active RA diagnosed for at least 8weeks but no longer than four years, according to the revised 1987,2000, or 2010 criteria for the classification of RA (American RheumatismAssociation or Americal College of Rheumatology).

Subjects with “early rheumatoid arthritis” are those subjects with RAdiagnosed for at least eight weeks but no longer than four years,according to the revised 1987, 2000, or 2010 criteria for classificationof RA (American Rheumatism Association or Americal College ofRheumatology). RA includes, for example, juvenile-onset RA, juvenileidiopathic arthritis (JIA), or juvenile RA (JRA).

Patients with “incipient RA” have early polyarthritis that does notfully meet ACR criteria for a diagnosis of RA, in association with thepresence of RA-specific prognostic biomarkers such as anti-CCP andshared epitope. They include patients with positive anti-CCP antibodieswho present with polyarthritis, but do not yet have a diagnosis of RA,and are at high risk for going on to develop bona fide ACR criteria RA(95% probability).

The term “inflammatory arthritis” encompasses any arthritis caused by anautoimmune condition. Nonlimiting examples of inflammatory arthritis andautoimmune conditions that may involve inflammatory arthritis includerheumatoid arthritis (including juvenile-onset RA, juvenile idiopathicarthritis (JIA), and juvenile rheumatoid arthritis (JRA)), ankylosingspondylitis, mixed connective tissue disease (MCTD), psoriaticarthritis, reactive arthritis, scleroderma, Still's disease, systemiclupus erythematosus, acute and chronic arthritis, rheumatoid synovitis,gout or gouty arthritis, acute immunological arthritis, chronicinflammatory arthritis, degenerative arthritis, type II collagen-inducedarthritis, infectious arthritis, septic arthritis, Lyme arthritis,proliferative arthritis, vertebral arthritis, osteoarthritis, arthritischronica progrediente, arthritis deformans, polyarthritis chronicaprimaria, reactive arthritis, menopausal arthritis, estrogen-depletionarthritis, Felty's syndrome, and rheumatic autoimmune disease other thanRA.

“Joint damage” is used in the broadest sense and refers to damage orpartial or complete destruction to any part of one or more joints,including the connective tissue and cartilage, where damage includesstructural and/or functional damage of any cause, and may or may notcause joint pain/arthalgia. It includes, without limitation, jointdamage associated with or resulting from inflammatory joint disease aswell as non-inflammatory joint disease. This damage may be caused by anycondition, such as an autoimmune disease, especially inflammatoryarthritis, and most especially rheumatoid arthritis. For purposesherein, joints are points of contact between elements of a skeleton (ofa vertebrate such as an animal) with the parts that surround and supportit and include, but are not limited to, for example, hips, jointsbetween the vertebrae of the spine, joints between the spine and pelvis(sacroiliac joints), joints where the tendons and ligaments attach tobones, joints between the ribs and spine, shoulders, knees, feet,elbows, hands, fingers, ankles and toes, but especially joints in thehands and feet.

The term “lupus” as used herein is an autoimmune disease or disorderthat in general involves antibodies that attack connective tissue. Theprincipal form of lupus is a systemic one, systemic lupus erythematosus(SLE), including cutaneous SLE and subacutecutaneous SLE, as well asother types of lupus (including nephritis, extrarenal, cerebritis,pediatric, non-renal, discoid, and alopecia). In certain embodiments,the term “systemic lupus erythematosus” refers to a chronic autoimmunedisease that can result in skin lesions, joint pain and swelling, kidneydisease (lupus nephritis), fluid around the heart and/or lungs,inflammation of the heart, and various other systemic conditions. Incertain embodiments, the term “lupus nephritis” refers to inflammationof the kidneys that occurs in patients with SLE. Lupus nephritis mayinclude, for example, glomerulonephritis and/or interstitial nephritis,and can lead to hypertension, proteinuria, and kidney failure. Lupusnephritis may be classified based on severity and extent of disease, forexample, as defined by the International Society ofNephrology/Renal/Pathology Society. Lupus nephritis classes includeclass I (minimal mesangial lupus nephritis), class II (mesangialproliferative lupus nephritis), class III (focal lupus nephritis), classIV (diffuse segmental (IV-S) or diffuse global (IV-G) lupus nephritis),class V (membranous lupus nephritis), and class VI (advanced sclerosinglupus nephritis). The term “lupus nephritis” encompasses all of theclasses.

The term “multiple sclerosis” (“MS”) refers to the chronic and oftendisabling disease of the central nervous system characterized by theprogressive destruction of the myelin. “Demyelination” occurs when themyelin sheath becomes inflamed, injured, and detaches from the nervefiber. There are four internationally recognized forms of MS, namely,primary progressive multiple sclerosis (PPMS), relapsing-remittingmultiple sclerosis (RRMS), secondary progressive multiple sclerosis(SPMS), and progressive relapsing multiple sclerosis (PRMS).

“Primary progressive multiple sclerosis” or “PPMS” is characterized by agradual progression of the disease from its onset with no superimposedrelapses and remissions at all. There may be periods of a leveling offof disease activity and there may be good and bad days or weeks. PPMSdiffers from RRMS and SPMS in that onset is typically in the latethirties or early forties, men are as likely as women to develop it, andinitial disease activity is often in the spinal cord and not in thebrain. PPMS often migrates into the brain, but is less likely to damagebrain areas than RRMS or SPMS; for example, people with PPMS are lesslikely to develop cognitive problems. PPMS is the sub-type of MS that isleast likely to show inflammatory (gadolinium enhancing) lesions on MRIscans. The primary progressive form of the disease affects between 10and 15% of all people with multiple sclerosis. PPMS may be definedaccording to the criteria in McDonald et al. Ann Neurol 50:121-7 (2001).The subject with PPMS treated herein is usually one with a probable ordefinitive diagnosis of PPMS.

“Relapsing-remitting multiple sclerosis” or “RRMS” is characterized byrelapses (also known as exacerbations) during which time new symptomscan appear and old ones resurface or worsen. The relapses are followedby periods of remission, during which time the person fully or partiallyrecovers from the deficits acquired during the relapse. Relapses canlast for days, weeks, or months, and recovery can be slow and gradual oralmost instantaneous. The vast majority of people presenting with MS arefirst diagnosed with RRMS. This is typically when they are in theirtwenties or thirties, though diagnoses much earlier or later are known.Twice as many women as men present with this sub-type of MS. Duringrelapses, myelin, a protective insulating sheath around the nerve fibers(neurons) in the white matter regions of the central nervous system(CNS), may be damaged in an inflammatory response by the body's ownimmune system. This causes a wide variety of neurological symptoms thatvary considerably depending on which areas of the CNS are damaged.Immediately after a relapse, the inflammatory response dies down and aspecial type of glial cell in the CNS (called an oligodendrocyte)sponsors remyelination—a process whereby the myelin sheath around theaxon may be repaired. It is this remyelination that may be responsiblefor the remission. Approximately 50% of patients with RRMS convert toSPMS within 10 years of disease onset. After 30 years, this figure risesto 90%. At any one time, the relapsing-remitting form of the diseaseaccounts around 55% of all people with MS.

“Secondary progressive multiple sclerosis” or “SPMS” is characterized bya steady progression of clinical neurological damage with or withoutsuperimposed relapses and minor remissions and plateau. People whodevelop SPMS will have previously experienced a period of RRMS which mayhave lasted anywhere from two to forty years or more. Any superimposedrelapses and remissions tend to tail off over time. From the onset ofthe secondary progressive phase of the disease, disability startsadvancing much quicker than it did during RRMS though the progress canstill be quite slow in some individuals. SPMS tends to be associatedwith lower levels of inflammatory lesion formation than in RRMS but thetotal burden of disease continues to progress. At any one time, SPMSaccounts around 30% of all people with multiple sclerosis.

“Progressive relapsing multiple sclerosis” or “PRMS” is characterized bya steady progression of clinical neurological damage with superimposedrelapses and remissions. There is significant recovery immediatelyfollowing a relapse but between relapses there is a gradual worsening ofsymptoms. PRMS affects around 5% of all people with multiple sclerosis.Some neurologists believe PRMS is a variant of PPMS.

The term “CD16+ disorder” means a disease in which CD16+ monocytes of amammal cause, mediate or otherwise contribute to morbidity in themammal. Also included are diseases in which reduction of CD16+ monocyteshas an ameliorative effect on progression of the disease. Includedwithin this term are CD16+ inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, etc. In some embodiments, CD16+inflammatory diseases include inflammatory diseases that are notresponsive to methotrexate therapy. In some embodiments, CD16+inflammatory diseases include methotrexate-resistant rheumatoidarthritis, methotrexate-resistant multiple sclerosis,methotrexate-resistant lupus, methotrexate-resistant inflammatory boweldisease, methotrexate-resistant Crohn's disease, methotrexate-resistantasthma, and methotrexate-resistant psoriasis. In certain embodiments,patients having methotrexate-resistant diseases, such asmethotrexate-resistant rheumatoid arthritis, are referred to asmethotrexate incomplete responders or methotrexate inadequateresponders. In some embodiments, a subject with a CD16+ disorder is amethotrexate inadequate responder. In some embodiments, patients havingTNF inhibitor-resistant diseases, such as TNF inhibitor-resistantrheumatoid arthritis, are referred to as TNF inhibitor incompleteresponders or TNF inhibitor inadequate responders. In some embodiments,a subject with a CD16+ disorder is a TNF inhibitor inadequate responder.

Examples of CD16+ disorders that can be treated according to theinvention include, but are not limited to, systemic lupus erythematosus,lupus nephritis, rheumatoid arthritis, juvenile chronic arthritis,juvenile idiopathic arthritis (JIA) (including systemic JIA andpolyarticular course JIA), psoriatic arthritis, polymyalgia rheumatic,osteoarthritis, adult-onset Still's disease, spondyloarthropathies,ankylosing spondylitis, systemic sclerosis (scleroderma), idiopathicinflammatory myopathies (dermatomyositis, polymyositis), Sjogren'ssyndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia(immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia), uveitis, thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis,chronic thyroiditis), diabetes mellitus, immune-mediated renal disease(glomerulonephritis, tubulointerstitial nephritis), nephritis (such asmesangium proliferative nephritis), osteoporosis, cachexia (includingcancerous cachexia), tumors, prostate cancer, choroidalneovascularization (such as age-related macular degeneration, idiopathicchoroidal neovascularization, cyopic choroidal neovascularization,idiopathic choroidal neovascularization), ocular inflammatory disease(e.g. panuveitis, anterior aveitis, intermediate uveitis, scleritis,keratitis, orbital inflammation, optic neuritis, dry eye, diabeticretinopathy, proliferative vitreoretinopathy, postoperativeinflammation), muscle atrophy, demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barre syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory bowel disease (IBD), including ulcerative colitis, Crohn'sdisease, gluten-sensitive enteropathy, and Whipple's disease,pancreatitis, islet transplantation (e.g., pancreatic islettransplantation), autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis, allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, delayed hypersensitivity, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonia, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, transplantation associated diseases including graftrejection and graft-versus-host-disease, chronic rejection; fibrosis,including kidney fibrosis and hepatic fibrosis, cardiovascular disease,including atherosclerosis and coronary artery disease, giant cellarteritis (GCA), Takayasu's arteritis (TA), arteritis nodosa,cardiovascular events associated with chronic kidney disease, myocardialinfarction, ischemia-induced severe arrhythmia, and congestive heartfailure, diabetes, including type II diabetes, Bronchiolitis obliteranswith organizing pneumonia (BOOP), hemophagocytic syndrome, macrophageactivation syndrome, sarcoidosis, and periodontitis. Infectious diseasesincluding viral diseases such as AIDS (HIV infection), hepatitis A, B,C, D, and E, herpes, etc., bacterial infections, fungal infections,protozoal infections and parasitic infections.

The term “methotrexate inadequate responder” as used herein refers to asubject who has experienced, or is experiencing, an inadequate responseto methotrexate treatment, for example, because of toxicity and/orinadequate efficacy at standard doses. In some embodiments, amethotrexate inadequate responder has experienced, or is experiencing,an inadequate response to methotrexate after receiving a standard dosefor at least two weeks, at least three weeks, at least four weeks, atleast six weeks, or at least twelve weeks.

The term “TNF inhibitor inadequate responder” as used herein refers to asubject who has experienced, or is experiencing, an inadequate responseto a TNF inhibitor, for example, because of toxicity and/or inadequateefficacy at standard doses. In some embodiments, a TNF inhibitorinadequate responder has experienced, or is experiencing, an inadequateresponse to a TNF inhibitor after receiving a standard dose for at leasttwo weeks, at least three weeks, at least four weeks, at least sixweeks, or at least twelve weeks. In some embodiments, a TNF inhibitorinadequate responder has experienced, or is experiencing, an inadequateresponse to a TNF inhibitor selected from infliximab, adalimumab,certolizumab pegol, golimumab, and etanercept. In some embodiments, theTNF inhibitor is a TNF-α inhibitor.

The term “substantially the same” and “substantially unchanged” andgrammatical variants thereof, when used to refer to the level of aprotein or cell type, such as CD16− monocytes, denote a sufficientlyhigh degree of similarity between the levels being compared, e.g., asindicated by numeric values, such that one of skill in the art wouldconsider the difference between the levels to be of little or nobiological and/or statistical significance.

The term “substantially reduced” and “substantially decreased” andgrammatical variants thereof, when used to refer to the level of aprotein or cell type, such as CD16+ monocytes, denote a sufficientlyhigh degree of difference between the levels being compared, e.g., asindicated by numeric values, such that one of skill in the art wouldconsider the difference between the levels to be of biological and/orstatistical significance.

An “anti-[factor] agent” or a “[factor] inhibitor” as used herein, referto an agent that antagonizes the factor activity, such as by binding tothe factor or a receptor for the factor (if any), or by specificallyinhibiting expression of the factor or a receptor for the factor (ifany). Exemplary anti-[factor] agents include, but are not limited to,anti-[factor] antibodies, anti-[factor] receptor antibodies, soluble[factor] receptors that bind to the factor, small molecules that bindthe [factor] or [factor] receptor, antisense oligonucleotides that arecomplementary to [factor] or [factor] receptor pre-mRNA or mRNA, etc.Nonlimiting exemplary factors include TNF-α, IL-1, IL-6, CD20, CD19, andGM-CSF.

An agent “antagonizes” factor activity when the agent neutralizes,blocks, inhibits, abrogates, reduces, and/or interferes with theactivity of the factor, including its binding to one or more receptorswhen the factor is a ligand.

“Treatment,” as used herein, refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) the targeted pathologic condition or disorder. Incertain embodiments, the term “treatment” covers any administration orapplication of a therapeutic for disease in a mammal, including a human,and includes inhibiting or slowing the disease or progression of thedisease; partially or fully relieving the disease, for example, bycausing regression, or restoring or repairing a lost, missing, ordefective function; stimulating an inefficient process; or causing thedisease plateau to have reduced severity. The term “treatment” alsoincludes reducing the severity of any phenotypic characteristic and/orreducing the incidence, degree, or likelihood of that characteristic.Those in need of treatment include those already with the disorder aswell as those prone to have the disorder or those in whom the disorderis to be prevented.

“Chronic” administration refers to administration of an agent in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

The term “effective amount” or “therapeutically effective amount” refersto an amount of a drug effective to treat a disease or disorder in asubject. In certain embodiments, an effective amount refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic or prophylactic result. A therapeutically effectiveamount of an anti-CSF1R antibody of the invention may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the anti-CSF1R antibody to elicit adesired response in the individual. A therapeutically effective amountencompasses an amount in which any toxic or detrimental effects of theanti-CSF1R antibody are outweighed by the therapeutically beneficialeffects. In some embodiments, the expression “effective amount” refersto an amount of the antibody that is effective for treating the CD16+disorder. When the disorder is RA, such effective amount can result inone or more of: reducing the signs or symptoms of RA (e.g. achievingACR20, ACR50, or ACR70 response at week 24 and/or week 48), reducingdisease activity (e.g. Disease Activity Score, DAS28), slowing theprogression of structural joint damage, improving physical function,etc.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, but not necessarily, since aprophylactic dose is used in subjects prior to or at an earlier stage ofdisease, the prophylactically effective amount would be less than thetherapeutically effective amount.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid, or liquid filler, diluent, encapsulating material,formulation auxiliary, or carrier conventional in the art for use with atherapeutic agent that together comprise a “pharmaceutical composition”for administration to a subject. A pharmaceutically acceptable carrieris non-toxic to recipients at the dosages and concentrations employedand is compatible with other ingredients of the formulation. Thepharmaceutically acceptable carrier is appropriate for the formulationemployed. For example, if the therapeutic agent is to be administeredorally, the carrier may be a gel capsule. If the therapeutic agent is tobe administered subcutaneously, the carrier ideally is not irritable tothe skin and does not cause injection site reaction.

Anti-CSF1R Antibodies

Anti-CSF1R antibodies include, but are not limited to, humanizedantibodies, chimeric antibodies, mouse antibodies, human antibodies, andantibodies comprising the heavy chain and/or light chain CDRs discussedherein.

Exemplary Humanized Antibodies

In some embodiments, humanized antibodies that bind CSF1R are provided.Humanized antibodies are useful as therapeutic molecules becausehumanized antibodies reduce or eliminate the human immune response tonon-human antibodies (such as the human anti-mouse antibody (HAMA)response), which can result in an immune response to an antibodytherapeutic, and decreased effectiveness of the therapeutic.

Nonlimiting exemplary humanized antibodies include huAb1 through huAb16,described herein. Nonlimiting exemplary humanized antibodies alsoinclude antibodies comprising a heavy chain variable region of anantibody selected from huAb1 to huAb16 and/or a light chain variableregion of an antibody selected from huAb1 to huAb16. Nonlimitingexemplary humanized antibodies include antibodies comprising a heavychain variable region selected from SEQ ID NOs: 39 to 45 and/or a lightchain variable region selected from SEQ ID NOs: 46 to 52. Exemplaryhumanized antibodies also include, but are not limited to, humanizedantibodies comprising heavy chain CDR1, CDR2, and CDR3, and/or lightchain CDR1, CDR2, and CDR3 of an antibody selected from 0301, 0302, and0311.

In some embodiments, a humanized anti-CSF1R antibody comprises heavychain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, and CDR3 ofan antibody selected from 0301, 0302, and 0311. Nonlimiting exemplaryhumanized anti-CSF1R antibodies include antibodies comprising sets ofheavy chain CDR1, CDR2, and CDR3 selected from: SEQ ID NOs: 15, 16, and17; SEQ ID NOs: 21, 22, and 23; and SEQ ID NOs: 27, 28, and 29.Nonlimiting exemplary humanized anti-CSF1R antibodies also includeantibodies comprising sets of light chain CDR1, CDR2, and CDR3 selectedfrom: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 24, 25, and 26; and SEQ IDNOs: 30, 31, and 32.

Nonlimiting exemplary humanized anti-CSF1R antibodies include antibodiescomprising the sets of heavy chain CDR1, CDR2, and CDR3, and light chainCDR1, CDR2, and CDR3 in Table 1 (SEQ ID NOs shown; see Table 8 forsequences). Each row of Table 1 shows the heavy chain CDR1, CDR2, andCDR3, and light chain CDR1, CDR2, and CDR3 of an exemplary antibody.

TABLE 1 Heavy chain and light chain CDRs Heavy chain Light chain CDR1CDR2 CDR3 CDR1 CDR2 CDR3 Ab SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID0301 15 16 17 18 19 20 0302 21 22 23 24 25 26 0311 27 28 29 30 31 32

Further Exemplary Humanized Antibodies

In some embodiments, a humanized anti-CSF1R antibody comprises a heavychain comprising a variable region sequence that is at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to asequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45, and whereinthe antibody binds CSF1R. In some embodiments, a humanized anti-CSF1Rantibody comprises a light chain comprising a variable region sequencethat is at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46to 52, wherein the antibody binds CSF1R. In some embodiments, ahumanized anti-CSF1R antibody comprises a heavy chain comprising avariable region sequence that is at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to a sequence selected fromSEQ ID NOs: 9, 11, 13, and 39 to 45; and a light chain comprising avariable region sequence that is at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to a sequence selected fromSEQ ID NOs: 10, 12, 14, and 46 to 52; wherein the antibody binds CSF1R.

As used herein, whether a particular polypeptide is, for example, atleast 95% identical to an amino acid sequence can be determined using,e.g., a computer program. When determining whether a particular sequenceis, for example, 95% identical to a reference sequence, the percentageof identity is calculated over the full length of the reference aminoacid sequence.

In some embodiments, a humanized anti-CSF1R antibody comprises at leastone of the CDRs discussed herein. That is, in some embodiments, ahumanized anti-CSF1R antibody comprises at least one CDR selected from aheavy chain CDR1 discussed herein, a heavy chain CDR2 discussed herein,a heavy chain CDR3 discussed herein, a light chain CDR1 discussedherein, a light chain CDR2 discussed herein, and a light chain CDR3discussed herein. Further, in some embodiments, a humanized anti-CSF1Rantibody comprises at least one mutated CDR based on a CDR discussedherein, wherein the mutated CDR comprises 1, 2, 3, or 4 amino acidsubstitutions relative to the CDR discussed herein. In some embodiments,one or more of the amino acid substitutions are conservative amino acidsubstitutions. One skilled in the art can select one or more suitableconservative amino acid substitutions for a particular CDR sequence,wherein the suitable conservative amino acid substitutions are notpredicted to significantly alter the binding properties of the antibodycomprising the mutated CDR.

Exemplary humanized anti-CSF1R antibodies also include antibodies thatcompete for binding to CSF1R with an antibody described herein. Thus, insome embodiments, a humanized anti-CSF1R antibody is provided thatcompetes for binding to CSF1R with an antibody selected from Fabs 0301,0302, and 0311; and bivalent (i.e., having two heavy chains and twolight chains) antibody versions of those Fabs.

Exemplary Humanized Antibody Constant Regions

In some embodiments, a humanized antibody described herein comprises oneor more human constant regions. In some embodiments, the human heavychain constant region is of an isotype selected from IgA, IgG, and IgD.In some embodiments, the human light chain constant region is of anisotype selected from κ and λ. In some embodiments, a humanized antibodydescribed herein comprises a human IgG constant region. In someembodiments, a humanized antibody described herein comprises a humanIgG4 heavy chain constant region. In some such embodiments, a humanizedantibody described herein comprises an S241P mutation in the human IgG4constant region. In some embodiments, a humanized antibody describedherein comprises a human IgG4 constant region and a human κ light chain.

The choice of heavy chain constant region can determine whether or notan antibody will have effector function in vivo. Such effector function,in some embodiments, includes antibody-dependent cell-mediatedcytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), andcan result in killing of the cell to which the antibody is bound. Insome methods of treatment, including methods of treating some cancers,cell killing may be desirable, for example, when the antibody binds to acell that supports the maintenance or growth of the tumor. Exemplarycells that may support the maintenance or growth of a tumor include, butare not limited to, tumor cells themselves, cells that aid in therecruitment of vasculature to the tumor, and cells that provide ligands,growth factors, or counter-receptors that support or promote tumorgrowth or tumor survival. In some embodiments, when effector function isdesirable, an anti-CSF1R antibody comprising a human IgG1 heavy chain ora human IgG3 heavy chain is selected.

In some methods of treatment, effector function may not be desirable.For example, in some embodiments, it may be desirable that antibodiesused in the treatment of lupus and/or MS and/or RA and/or osteolysis donot have effector function. Thus, in some embodiments, anti-CSF1Rantibodies developed for the treatment of cancer may not be suitable foruse in treatment of lupus and/or MS and/or RA and/or osteolysis.Accordingly, in some embodiments, an anti-CSF1R antibody that lackssignificant effector function is used in treatment of lupus and/or MSand/or RA and/or osteolysis. In some embodiments, an anti-CSF1R antibodyfor treatment of lupus and/or MS and/or RA and/or osteolysis comprises ahuman IgG4 or IgG2 heavy chain constant region. In some embodiments, anIgG4 constant region comprises an S241P mutation.

An antibody may be humanized by any method. Nonlimiting exemplarymethods of humanization include methods described, e.g., in U.S. Pat.Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370; Jones etal., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-27(1988); Verhoeyen et al., Science 239: 1534-36 (1988); and U.S.Publication No. US 2009/0136500.

As noted above, a humanized antibody is an antibody in which at leastone amino acid in a framework region of a non-human variable region hasbeen replaced with the amino acid from the corresponding location in ahuman framework region. In some embodiments, at least two, at leastthree, at least four, at least five, at least six, at least seven, atleast eight, at least nine, at least 10, at least 11, at least 12, atleast 15, or at least 20 amino acids in the framework regions of anon-human variable region are replaced with an amino acid from one ormore corresponding locations in one or more human framework regions.

In some embodiments, some of the corresponding human amino acids usedfor substitution are from the framework regions of different humanimmunoglobulin genes. That is, in some such embodiments, one or more ofthe non-human amino acids may be replaced with corresponding amino acidsfrom a human framework region of a first human antibody or encoded by afirst human immunoglobulin gene, one or more of the non-human aminoacids may be replaced with corresponding amino acids from a humanframework region of a second human antibody or encoded by a second humanimmunoglobulin gene, one or more of the non-human amino acids may bereplaced with corresponding amino acids from a human framework region ofa third human antibody or encoded by a third human immunoglobulin gene,etc. Further, in some embodiments, all of the corresponding human aminoacids being used for substitution in a single framework region, forexample, FR2, need not be from the same human framework. In someembodiments, however, all of the corresponding human amino acids beingused for substitution are from the same human antibody or encoded by thesame human immunoglobulin gene.

In some embodiments, an antibody is humanized by replacing one or moreentire framework regions with corresponding human framework regions. Insome embodiments, a human framework region is selected that has thehighest level of homology to the non-human framework region beingreplaced. In some embodiments, such a humanized antibody is aCDR-grafted antibody.

In some embodiments, following CDR-grafting, one or more framework aminoacids are changed back to the corresponding amino acid in a mouseframework region. Such “back mutations” are made, in some embodiments,to retain one or more mouse framework amino acids that appear tocontribute to the structure of one or more of the CDRs and/or that maybe involved in antigen contacts and/or appear to be involved in theoverall structural integrity of the antibody. In some embodiments, tenor fewer, nine or fewer, eight or fewer, seven or fewer, six or fewer,five or fewer, four or fewer, three or fewer, two or fewer, one, or zeroback mutations are made to the framework regions of an antibodyfollowing CDR grafting.

In some embodiments, a humanized antibody also comprises a human heavychain constant region and/or a human light chain constant region.

Exemplary Chimeric Antibodies

In some embodiments, an anti-CSF1R antibody is a chimeric antibody. Insome embodiments, an anti-CSF1R antibody comprises at least onenon-human variable region and at least one human constant region. Insome such embodiments, all of the variable regions of an anti-CSF1Rantibody are non-human variable regions, and all of the constant regionsof an anti-CSF1R antibody are human constant regions. In someembodiments, one or more variable regions of a chimeric antibody aremouse variable regions. The human constant region of a chimeric antibodyneed not be of the same isotype as the non-human constant region, ifany, it replaces. Chimeric antibodies are discussed, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA 81:6851-55 (1984).

Nonlimiting exemplary chimeric antibodies include chimeric antibodiescomprising the heavy and/or light chain variable regions of an antibodyselected from 0301, 0302, and 0311. Additional nonlimiting exemplarychimeric antibodies include chimeric antibodies comprising heavy chainCDR1, CDR2, and CDR3, and/or light chain CDR1, CDR2, and CDR3 of anantibody selected from 0301, 0302, and 0311.

Nonlimiting exemplary chimeric anti-CSF1R antibodies include antibodiescomprising the following pairs of heavy and light chain variableregions: SEQ ID NOs: 9 and 10; SEQ ID NOs: 11 and 12; and SEQ ID NOs: 13and 14.

Nonlimiting exemplary anti-CSF1R antibodies include antibodiescomprising a set of heavy chain CDR1, CDR2, and CDR3, and light chainCDR1, CDR2, and CDR3 shown above in Table 1.

Further Exemplary Chimeric Antibodies

In some embodiments, a chimeric anti-CSF1R antibody comprises a heavychain comprising a variable region sequence that is at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to asequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45, wherein theantibody binds CSF1R. In some embodiments, a chimeric anti-CSF1Rantibody comprises a light chain comprising a variable region sequencethat is at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46to 52, wherein the antibody binds CSF1R. In some embodiments, a chimericanti-CSF1R antibody comprises a heavy chain comprising a variable regionsequence that is at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identical to a sequence selected from SEQ ID NOs: 9, 11,13, and 39 to 45; and a light chain comprising a variable regionsequence that is at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identical to a sequence selected from SEQ ID NOs: 10, 12,14, and 46 to 52; wherein the antibody binds CSF1R.

In some embodiments, a chimeric anti-CSF1R antibody comprises at leastone of the CDRs discussed herein. That is, in some embodiments, achimeric anti-CSF1R antibody comprises at least one CDR selected from aheavy chain CDR1 discussed herein, a heavy chain CDR2 discussed herein,a heavy chain CDR3 discussed herein, a light chain CDR1 discussedherein, a light chain CDR2 discussed herein, and a light chain CDR3discussed herein. Further, in some embodiments, a chimeric anti-CSF1Rantibody comprises at least one mutated CDR based on a CDR discussedherein, wherein the mutated CDR comprises 1, 2, 3, or 4 amino acidsubstitutions relative to the CDR discussed herein. In some embodiments,one or more of the amino acid substitutions are conservative amino acidsubstitutions. One skilled in the art can select one or more suitableconservative amino acid substitutions for a particular CDR sequence,wherein the suitable conservative amino acid substitutions are notpredicted to significantly alter the binding properties of the antibodycomprising the mutated CDR.

Exemplary chimeric anti-CSF1R antibodies also include chimericantibodies that compete for binding to CSF1R with an antibody describedherein. Thus, in some embodiments, a chimeric anti-CSF1R antibody isprovided that competes for binding to CSF1R with an antibody selectedfrom Fabs 0301, 0302, and 0311; and bivalent (i.e., having two heavychains and two light chains) antibody versions of those Fabs.

Exemplary Chimeric Antibody Constant Regions

In some embodiments, a chimeric antibody described herein comprises oneor more human constant regions. In some embodiments, the human heavychain constant region is of an isotype selected from IgA, IgG, and IgD.In some embodiments, the human light chain constant region is of anisotype selected from κ and λ. In some embodiments, a chimeric antibodydescribed herein comprises a human IgG constant region. In someembodiments, a chimeric antibody described herein comprises a human IgG4heavy chain constant region. In some such embodiments, a chimericantibody described herein comprises an S241P mutation in the human IgG4constant region. In some embodiments, a chimeric antibody describedherein comprises a human IgG4 constant region and a human κ light chain.

As noted above, whether or not effector function is desirable may dependon the particular method of treatment intended for an antibody. Thus, insome embodiments, when effector function is desirable, a chimericanti-CSF1R antibody comprising a human IgG1 heavy chain constant regionor a human IgG3 heavy chain constant region is selected. In someembodiments, when effector function is not desirable, a chimericanti-CSF1R antibody comprising a human IgG4 or IgG2 heavy chain constantregion is selected.

Exemplary Human Antibodies

Human antibodies can be made by any suitable method. Nonlimitingexemplary methods include making human antibodies in transgenic micethat comprise human immunoglobulin loci. See, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA 90: 2551-55 (1993); Jakobovits et al., Nature362: 255-8 (1993); Lonberg et al., Nature 368: 856-9 (1994); and U.S.Pat. Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807;6,300,129; 6,255,458; 5,877,397; 5,874,299; and 5,545,806.

Nonlimiting exemplary methods also include making human antibodies usingphage display libraries. See, e.g., Hoogenboom et al., J. Mol. Biol.227: 381-8 (1992); Marks et al., J. Mol. Biol. 222: 581-97 (1991); andPCT Publication No. WO 99/10494.

In some embodiments, a human anti-CSF1R antibody binds to a polypeptidehaving the sequence of SEQ ID NO: 1. Exemplary human anti-CSF1Rantibodies also include antibodies that compete for binding to CSF1Rwith an antibody described herein. Thus, in some embodiments, a humananti-CSF1R antibody is provided that competes for binding to CSF1R withan antibody selected from Fabs 0301, 0302, and 0311, and bivalent (i.e.,having two heavy chains and two light chains) antibody versions of thoseFabs.

In some embodiments, a human anti-CSF1R antibody comprises one or morehuman constant regions. In some embodiments, the human heavy chainconstant region is of an isotype selected from IgA, IgG, and IgD. Insome embodiments, the human light chain constant region is of an isotypeselected from κ and λ. In some embodiments, a human antibody describedherein comprises a human IgG constant region. In some embodiments, ahuman antibody described herein comprises a human IgG4 heavy chainconstant region. In some such embodiments, a human antibody describedherein comprises an S241P mutation in the human IgG4 constant region. Insome embodiments, a human antibody described herein comprises a humanIgG4 constant region and a human κ light chain.

In some embodiments, when effector function is desirable, a humananti-CSF1R antibody comprising a human IgG1 heavy chain constant regionor a human IgG3 heavy chain constant region is selected. In someembodiments, when effector function is not desirable, a human anti-CSF1Rantibody comprising a human IgG4 or IgG2 heavy chain constant region isselected.

Additional Exemplary Anti-CSF1R Antibodies

Exemplary anti-CSF1R antibodies also include, but are not limited to,mouse, humanized, human, chimeric, and engineered antibodies thatcomprise, for example, one or more of the CDR sequences describedherein. In some embodiments, an anti-CSF1R antibody comprises a heavychain variable region described herein. In some embodiments, ananti-CSF1R antibody comprises a light chain variable region describedherein. In some embodiments, an anti-CSF1R antibody comprises a heavychain variable region described herein and a light chain variable regiondescribed herein. In some embodiments, an anti-CSF1R antibody comprisesheavy chain CDR1, CDR2, and CDR3 described herein. In some embodiments,an anti-CSF1R antibody comprises light chain CDR1, CDR2, and CDR3described herein. In some embodiments, an anti-CSF1R antibody comprisesheavy chain CDR1, CDR2, and CDR3 described herein and light chain CDR1,CDR2, and CDR3 described herein.

In some embodiments, an anti-CSF1R antibody comprises a heavy chainvariable region of an antibody selected from Fabs 0301, 0302, and 0311.Nonlimiting exemplary anti-CSF1R antibodies also include antibodiescomprising a heavy chain variable region of an antibody selected fromhumanized antibodies huAb1 to huAb16. Nonlimiting exemplary anti-CSF1Rantibodies include antibodies comprising a heavy chain variable regioncomprising a sequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45.

In some embodiments, an anti-CSF1R antibody comprises a light chainvariable region of an antibody selected from Fabs 0301, 0302, and 0311.Nonlimiting exemplary anti-CSF1R antibodies also include antibodiescomprising a light chain variable region of an antibody selected fromhumanized antibodies huAb1 to huAb16. Nonlimiting exemplary anti-CSF1Rantibodies include antibodies comprising a light chain variable regioncomprising a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to52.

In some embodiments, an anti-CSF1R antibody comprises a heavy chainvariable region and a light chain variable region of an antibodyselected from Fabs 0301, 0302, and 0311. Nonlimiting exemplaryanti-CSF1R antibodies also include antibodies comprising a heavy chainvariable region and a light chain variable region of an antibodyselected from humanized antibodies huAb1 to huAb16. Nonlimitingexemplary anti-CSF1R antibodies include antibodies comprising thefollowing pairs of heavy and light chain variable regions: SEQ ID NOs: 9and 10; SEQ ID NOs: 11 and 12; and SEQ ID NOs: 13 and 14; SEQ ID NOs: 39and 40; SEQ ID NOs: 41 and 42; SEQ ID NOs: 43 and 44; SEQ ID NOs: 45 and46; SEQ ID NOs: 47 and 48; SEQ ID NOs: 49 and 50; and SEQ ID NOs: 51 and52. Nonlimiting exemplary anti-CSF1R antibodies also include antibodiescomprising the following pairs of heavy and light chains: SEQ ID NOs: 33and 34; SEQ ID NOs: 35 and 36; and SEQ ID NOs: 37 and 38.

In some embodiments, an anti-CSF1R antibody comprises heavy chain CDR1,CDR2, and CDR3 of an antibody selected from Fabs 0301, 0302, and 0311.Nonlimiting exemplary anti-CSF1R antibodies include antibodiescomprising sets of heavy chain CDR1, CDR2, and CDR3 selected from: SEQID NOs: 15, 16, and 17; SEQ ID NOs: 21, 22, and 23; and SEQ ID NOs: 27,28, and 29.

In some embodiments, an anti-CSF1R antibody comprises light chain CDR1,CDR2, and CDR3 of an antibody selected from Fabs 0301, 0302, and 0311.Nonlimiting exemplary anti-CSF1R antibodies include antibodiescomprising sets of light chain CDR1, CDR2, and CDR3 selected from: SEQID NOs: 18, 19, and 20; SEQ ID NOs: 24, 25, and 26; and SEQ ID NOs: 30,31, and 32.

In some embodiments, an anti-CSF1R antibody comprises heavy chain CDR1,CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 of an antibodyselected from Fabs 0301, 0302, and 0311.

Nonlimiting exemplary anti-CSF1R antibodies include antibodiescomprising the sets of heavy chain CDR1, CDR2, and CDR3, and light chainCDR1, CDR2, and CDR3 shown above in Table 1.

Further Exemplary Antibodies

In some embodiments, an anti-CSF1R antibody comprises a heavy chaincomprising a variable region sequence that is at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to a sequenceselected from SEQ ID NOs: 9, 11, 13, and 39 to 45, wherein the antibodybinds CSF1R. In some embodiments, an anti-CSF1R antibody comprises alight chain comprising a variable region sequence that is at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to asequence selected from SEQ ID NOs: 10, 12, 14, and 46 to 52, wherein theantibody binds CSF1R. In some embodiments, an anti-CSF1R antibodycomprises a heavy chain comprising a variable region sequence that is atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to a sequence selected from SEQ ID NOs: 9, 11, 13, and 39 to45; and a light chain comprising a variable region sequence that is atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to52; wherein the antibody binds CSF1R.

In some embodiments, an anti-CSF1R antibody comprises at least one ofthe CDRs discussed herein. That is, in some embodiments, an anti-CSF1Rantibody comprises at least one CDR selected from a heavy chain CDR1discussed herein, a heavy chain CDR2 discussed herein, a heavy chainCDR3 discussed herein, a light chain CDR1 discussed herein, a lightchain CDR2 discussed herein, and a light chain CDR3 discussed herein.Further, in some embodiments, an anti-CSF1R antibody comprises at leastone mutated CDR based on a CDR discussed herein, wherein the mutated CDRcomprises 1, 2, 3, or 4 amino acid substitutions relative to the CDRdiscussed herein. In some embodiments, one or more of the amino acidsubstitutions are conservative amino acid substitutions. One skilled inthe art can select one or more suitable conservative amino acidsubstitutions for a particular CDR sequence, wherein the suitableconservative amino acid substitutions are not predicted to significantlyalter the binding properties of the antibody comprising the mutated CDR.

Exemplary anti-CSF1R antibodies also include antibodies that compete forbinding to CSF1R with an antibody described herein. Thus, in someembodiments, an anti-CSF1R antibody is provided that competes forbinding to CSF1R with an antibody selected from Fabs 0301, 0302, and0311, and bivalent (i.e., having two heavy chains and two light chains)antibody versions of those Fabs.

Exemplary Antibody Constant Regions

In some embodiments, an antibody described herein comprises one or morehuman constant regions. In some embodiments, the human heavy chainconstant region is of an isotype selected from IgA, IgG, and IgD. Insome embodiments, the human light chain constant region is of an isotypeselected from κ and λ. In some embodiments, an antibody described hereincomprises a human IgG constant region. In some embodiments, an antibodydescribed herein comprises a human IgG4 heavy chain constant region. Insome such embodiments, an antibody described herein comprises an S241Pmutation in the human IgG4 constant region. In some embodiments, anantibody described herein comprises a human IgG4 constant region and ahuman κ light chain.

As noted above, whether or not effector function is desirable may dependon the particular method of treatment intended for an antibody. Thus, insome embodiments, when effector function is desirable, an anti-CSF1Rantibody comprising a human IgG1 heavy chain constant region or a humanIgG3 heavy chain constant region is selected. In some embodiments, wheneffector function is not desirable, an anti-CSF1R antibody comprising ahuman IgG4 or IgG2 heavy chain constant region is selected.

Exemplary Anti-CSF1R Heavy Chain Variable Regions

In some embodiments, anti-CSF1R antibody heavy chain variable regionsare provided. In some embodiments, an anti-CSF1R antibody heavy chainvariable region is a mouse variable region, a human variable region, ora humanized variable region.

An anti-CSF1R antibody heavy chain variable region comprises a heavychain CDR1, FR2, CDR2, FR3, and CDR3. In some embodiments, an anti-CSF1Rantibody heavy chain variable region further comprises a heavy chain FR1and/or FR4. Nonlimiting exemplary heavy chain variable regions include,but are not limited to, heavy chain variable regions having an aminoacid sequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45.

In some embodiments, an anti-CSF1R antibody heavy chain variable regioncomprises a CDR1 comprising a sequence selected from SEQ ID NOs: 15, 21,and 27.

In some embodiments, an anti-CSF1R antibody heavy chain variable regioncomprises a CDR2 comprising a sequence selected from SEQ ID NOs: 16, 22,and 28.

In some embodiments, an anti-CSF1R antibody heavy chain variable regioncomprises a CDR3 comprising a sequence selected from SEQ ID NOs: 17, 23,and 29.

Nonlimiting exemplary heavy chain variable regions include, but are notlimited to, heavy chain variable regions comprising sets of CDR1, CDR2,and CDR3 selected from: SEQ ID NOs: 15, 16, and 17; SEQ ID NOs: 21, 22,and 23; and SEQ ID NOs: 27, 28, and 29.

In some embodiments, an anti-CSF1R antibody heavy chain comprises avariable region sequence that is at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to a sequence selected fromSEQ ID NOs: 9, 11, 13, and 39 to 45, wherein the heavy chain, togetherwith a light chain, is capable of forming an antibody that binds CSF1R.

In some embodiments, an anti-CSF1R antibody heavy chain comprises atleast one of the CDRs discussed herein. That is, in some embodiments, ananti-CSF1R antibody heavy chain comprises at least one CDR selected froma heavy chain CDR1 discussed herein, a heavy chain CDR2 discussedherein, and a heavy chain CDR3 discussed herein. Further, in someembodiments, an anti-CSF1R antibody heavy chain comprises at least onemutated CDR based on a CDR discussed herein, wherein the mutated CDRcomprises 1, 2, 3, or 4 amino acid substitutions relative to the CDRdiscussed herein. In some embodiments, one or more of the amino acidsubstitutions are conservative amino acid substitutions. One skilled inthe art can select one or more suitable conservative amino acidsubstitutions for a particular CDR sequence, wherein the suitableconservative amino acid substitutions are not predicted to significantlyalter the binding properties of the heavy chain comprising the mutatedCDR.

In some embodiments, a heavy chain comprises a heavy chain constantregion. In some embodiments, a heavy chain comprises a human heavy chainconstant region. In some embodiments, the human heavy chain constantregion is of an isotype selected from IgA, IgG, and IgD. In someembodiments, the human heavy chain constant region is an IgG constantregion. In some embodiments, a heavy chain comprises a human igG4 heavychain constant region. In some such embodiments, the human IgG4 heavychain constant region comprises an S241P mutation.

In some embodiments, when effector function is desirable, a heavy chaincomprises a human IgG1 or IgG3 heavy chain constant region. In someembodiments, when effector function is less desirable, a heavy chaincomprises a human IgG4 or IgG2 heavy chain constant region.

Exemplary Anti-CSF1R Light Chain Variable Regions

In some embodiments, anti-CSF1R antibody light chain variable regionsare provided. In some embodiments, an anti-CSF1R antibody light chainvariable region is a mouse variable region, a human variable region, ora humanized variable region.

An anti-CSF1R antibody light chain variable region comprises a lightchain CDR1, FR2, CDR2, FR3, and CDR3. In some embodiments, an anti-CSF1Rantibody light chain variable region further comprises a light chain FR1and/or FR4. Nonlimiting exemplary light chain variable regions includelight chain variable regions having an amino acid sequence selected fromSEQ ID NOs: 10, 12, 14, and 46 to 52.

In some embodiments, an anti-CSF1R antibody light chain variable regioncomprises a CDR1 comprising a sequence selected from SEQ ID NOs: 18, 24and 30.

In some embodiments, an anti-CSF1R antibody light chain variable regioncomprises a CDR2 comprising a sequence selected from SEQ ID NOs: 19, 25,and 31.

In some embodiments, an anti-CSF1R antibody light chain variable regioncomprises a CDR3 comprising a sequence selected from SEQ ID NOs: 20, 26,and 32.

Nonlimiting exemplary light chain variable regions include, but are notlimited to, light chain variable regions comprising sets of CDR1, CDR2,and CDR3 selected from: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 24, 25,and 26; and SEQ ID NOs: 30, 31, and 32.

In some embodiments, an anti-CSF1R antibody light chain comprises avariable region sequence that is at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to a sequence selected fromSEQ ID NOs: 10, 12, 14, and 46 to 52, wherein the light chain, togetherwith a heavy chain, is capable of forming an antibody that binds CSF1R.

In some embodiments, an anti-CSF1R antibody light chain comprises atleast one of the CDRs discussed herein. That is, in some embodiments, ananti-CSF1R antibody light chain comprises at least one CDR selected froma light chain CDR1 discussed herein, a light chain CDR2 discussedherein, and a light chain CDR3 discussed herein. Further, in someembodiments, an anti-CSF1R antibody light chain comprises at least onemutated CDR based on a CDR discussed herein, wherein the mutated CDRcomprises 1, 2, 3, or 4 amino acid substitutions relative to the CDRdiscussed herein. In some embodiments, one or more of the amino acidsubstitutions are conservative amino acid substitutions. One skilled inthe art can select one or more suitable conservative amino acidsubstitutions for a particular CDR sequence, wherein the suitableconservative amino acid substitutions are not predicted to significantlyalter the binding properties of the light chain comprising the mutatedCDR.

In some embodiments, a light chain comprises a human light chainconstant region. In some embodiments, a human light chain constantregion is selected from a human κ and a human λ light chain constantregion.

Exemplary Additional CSF1R Binding Molecules

In some embodiments, additional molecules that bind CSF1R are provided.Such molecules include, but are not limited to, non-canonical scaffolds,such as anti-calins, adnectins, ankyrin repeats, etc. See, e.g., Hosseet al., Prot. Sci. 15:14 (2006); Fiedler, M. and Skerra, A.,“Non-Antibody Scaffolds,” pp. 467-499 in Handbook of TherapeuticAntibodies, Dubel, S., ed., Wiley-VCH, Weinheim, Germany, 2007.

Exemplary Properties of Anti-CSF1R Antibodies

In some embodiments, an antibody having a structure described abovebinds to the CSF1R with a binding affinity (K_(D)) of less than 1 nM,blocks binding of CSF1 and/or IL-34 to CSF1R, and inhibits CSF1Rphosphorylation induced by CSF1 and/or IL-34.

In some embodiments, an anti-CSF1R antibody binds to the extracellulardomain of CSF1R (CSF1R-ECD). In some embodiments, an anti-CSF1R antibodyhas a binding affinity (K_(D)) for CSF1R of less than 1 nM, less than0.5 nM, less than 0.1 nM, or less than 0.05 nM. In some embodiments, ananti-CSF1R antibody has a K_(D) of between 0.01 and 1 nM, between 0.01and 0.5 nM, between 0.01 and 0.1 nM, between 0.01 and 0.05 nM, orbetween 0.02 and 0.05 nM.

In some embodiments, an anti-CSF1R antibody blocks ligand binding toCSF1R. In some embodiments, an anti-CSF1R antibody blocks binding ofCSF1 to CSF1R. In some embodiments, an anti-CSF1R antibody blocksbinding of IL-34 to CSF1R. In some embodiments, an anti-CSF1R antibodyblocks binding of both CSF1 and IL-34 to CSF1R. In some embodiments, anantibody that blocks ligand binding binds to the extracellular domain ofCSF1R. In some embodiments, an antibody blocks ligand binding to CSF1Rwhen it reduces the amount of detectable binding of a ligand to CSF1R byat least 50%, using the assay described, e.g., U.S. Pat. No. 8,206,715B2, Example 7, which is incorporated herein by reference for anypurpose. In some embodiments, an antibody reduces the amount ofdetectable binding of a ligand to CSF1R by at least 60%, at least 70%,at least 80%, or at least 90%. In some such embodiments, the antibody issaid to block ligand binding by at least 50%, at least 60%, at least70%, etc.

In some embodiments, an anti-CSF1R antibody inhibits ligand-inducedCSF1R phosphorylation. In some embodiments, an anti-CSF1R antibodyinhibits CSF1-induced CSF1R phosphorylation. In some embodiments, ananti-CSF1R antibody inhibits IL-34-induced CSF1R phosphorylation. Insome embodiments, an anti-CSF1R antibody inhibits both CSF1-induced andIL-34-induced CSF1R phosphorylation. In some embodiments, an antibody isconsidered to “inhibit ligand-induced CSF1R phosphorylation” when itreduces the amount of detectable ligand-induced CSF1R phosphorylation byat least 50%, using the assay described, e.g., U.S. Pat. No. 8,206,715B2, Example 6, which is incorporated herein by reference for anypurpose. In some embodiments, an antibody reduces the amount ofdetectable ligand-induced CSF1R phosphorylation by at least 60%, atleast 70%, at least 80%, or at least 90%. In some such embodiments, theantibody is said to inhibit ligand-induced CSF1R phosphorylation by atleast at least 50%, at least 60%, at least 70%, etc.

In some embodiments, an antibody inhibits monocyte proliferation and/orsurvival responses in the presence of CSF1 and/or IL-34. In someembodiments, an antibody is considered to “inhibit monocyteproliferation and/or survival responses” when it reduces the amount ofmonocyte proliferation and/or survival responses in the presence of CSF1and/or IL-34 by at least 50%, using the assay described, e.g., U.S. Pat.No. 8,206,715 B2, Example 10, which is incorporated herein by referencefor any purpose. In some embodiments, an antibody reduces the amount ofmonocyte proliferation and/or survival responses in the presence of CSF1and/or IL-34 by at least 60%, at least 70%, at least 80%, or at least90%. In some such embodiments, the antibody is said to inhibit monocyteproliferation and/or survival responses by at least at least 50%, atleast 60%, at least 70%, etc.

Exemplary Antibody Conjugates

In some embodiments, an anti-CSF1R antibody is conjugated to a labeland/or a cytotoxic agent. As used herein, a label is a moiety thatfacilitates detection of the antibody and/or facilitates detection of amolecule to which the antibody binds. Nonlimiting exemplary labelsinclude, but are not limited to, radioisotopes, fluorescent groups,enzymatic groups, chemiluminescent groups, biotin, epitope tags,metal-binding tags, etc. One skilled in the art can select a suitablelabel according to the intended application.

As used herein, a cytotoxic agent is a moiety that reduces theproliferative capacity of one or more cells. A cell has reducedproliferative capacity when the cell becomes less able to proliferate,for example, because the cell undergoes apoptosis or otherwise dies, thecell fails to proceed through the cell cycle and/or fails to divide, thecell differentiates, etc. Nonlimiting exemplary cytotoxic agentsinclude, but are not limited to, radioisotopes, toxins, andchemotherapeutic agents. One skilled in the art can select a suitablecytotoxic according to the intended application.

In some embodiments, a label and/or a cytotoxic agent is conjugated toan antibody using chemical methods in vitro. Nonlimiting exemplarychemical methods of conjugation are known in the art, and includeservices, methods and/or reagents commercially available from, e.g.,Thermo Scientific Life Science Research Produces (formerly Pierce;Rockford, Ill.), Prozyme (Hayward, Calif.), SACRI Antibody Services(Calgary, Canada), AbD Serotec (Raleigh, N.C.), etc. In someembodiments, when a label and/or cytotoxic agent is a polypeptide, thelabel and/or cytotoxic agent can be expressed from the same expressionvector with at least one antibody chain to produce a polypeptidecomprising the label and/or cytotoxic agent fused to an antibody chain.One skilled in the art can select a suitable method for conjugating alabel and/or cytotoxic agent to an antibody according to the intendedapplication.

Exemplary Leader Sequences

In order for some secreted proteins to express and secrete in largequantities, a leader sequence from a heterologous protein may bedesirable. In some embodiments, a leader sequence is selected from SEQID NOs: 3 and 4, which are light chain and heavy chain leader sequences,respectively. In some embodiments, employing heterologous leadersequences may be advantageous in that a resulting mature polypeptide mayremain unaltered as the leader sequence is removed in the ER during thesecretion process. The addition of a heterologous leader sequence may berequired to express and secrete some proteins.

Certain exemplary leader sequence sequences are described, e.g., in theonline Leader sequence Database maintained by the Department ofBiochemistry, National University of Singapore. See Choo et al., BMCBioinformatics, 6: 249 (2005); and PCT Publication No. WO 2006/081430.

Nucleic Acid Molecules Encoding Anti-CSF1R Antibodies

Nucleic acid molecules comprising polynucleotides that encode one ormore chains of anti-CSF1R antibodies are provided. In some embodiments,a nucleic acid molecule comprises a polynucleotide that encodes a heavychain or a light chain of an anti-CSF1R antibody. In some embodiments, anucleic acid molecule comprises both a polynucleotide that encodes aheavy chain and a polynucleotide that encodes a light chain, of ananti-CSF1R antibody. In some embodiments, a first nucleic acid moleculecomprises a first polynucleotide that encodes a heavy chain and a secondnucleic acid molecule comprises a second polynucleotide that encodes alight chain.

In some such embodiments, the heavy chain and the light chain areexpressed from one nucleic acid molecule, or from two separate nucleicacid molecules, as two separate polypeptides. In some embodiments, suchas when an antibody is an scFv, a single polynucleotide encodes a singlepolypeptide comprising both a heavy chain and a light chain linkedtogether.

In some embodiments, a polynucleotide encoding a heavy chain or lightchain of an anti-CSF1R antibody comprises a nucleotide sequence thatencodes a leader sequence, which, when translated, is located at the Nterminus of the heavy chain or light chain. As discussed above, theleader sequence may be the native heavy or light chain leader sequence,or may be another heterologous leader sequence.

Nucleic acid molecules may be constructed using recombinant DNAtechniques conventional in the art. In some embodiments, a nucleic acidmolecule is an expression vector that is suitable for expression in aselected host cell.

Anti-CSF1R Antibody Expression and Production

Vectors

Vectors comprising polynucleotides that encode anti-CSF1R heavy chainsand/or anti-CSF1R light chains are provided. Vectors comprisingpolynucleotides that encode anti-CSF1R heavy chains and/or anti-CSF1Rlight chains are also provided. Such vectors include, but are notlimited to, DNA vectors, phage vectors, viral vectors, retroviralvectors, etc. In some embodiments, a vector comprises a firstpolynucleotide sequence encoding a heavy chain and a secondpolynucleotide sequence encoding a light chain. In some embodiments, theheavy chain and light chain are expressed from the vector as twoseparate polypeptides. In some embodiments, the heavy chain and lightchain are expressed as part of a single polypeptide, such as, forexample, when the antibody is an scFv.

In some embodiments, a first vector comprises a polynucleotide thatencodes a heavy chain and a second vector comprises a polynucleotidethat encodes a light chain. In some embodiments, the first vector andsecond vector are transfected into host cells in similar amounts (suchas similar molar amounts or similar mass amounts). In some embodiments,a mole- or mass-ratio of between 5:1 and 1:5 of the first vector and thesecond vector is transfected into host cells. In some embodiments, amass ratio of between 1:1 and 1:5 for the vector encoding the heavychain and the vector encoding the light chain is used. In someembodiments, a mass ratio of 1:2 for the vector encoding the heavy chainand the vector encoding the light chain is used.

In some embodiments, a vector is selected that is optimized forexpression of polypeptides in CHO or CHO-derived cells, or in NSO cells.Exemplary such vectors are described, e.g., in Running Deer et al.,Biotechnol. Prog. 20:880-889 (2004).

In some embodiments, a vector is chosen for in vivo expression ofanti-CSF1R heavy chains and/or anti-CSF1R light chains in animals,including humans. In some such embodiments, expression of thepolypeptide is under the control of a promoter that functions in atissue-specific manner. For example, liver-specific promoters aredescribed, e.g., in PCT Publication No. WO 2006/076288.

Host Cells

In various embodiments, anti-CSF1R heavy chains and/or anti-CSF1R lightchains may be expressed in prokaryotic cells, such as bacterial cells;or in eukaryotic cells, such as fungal cells (such as yeast), plantcells, insect cells, and mammalian cells. Such expression may be carriedout, for example, according to procedures known in the art. Exemplaryeukaryotic cells that may be used to express polypeptides include, butare not limited to, COS cells, including COS 7 cells; 293 cells,including 293-6E cells; CHO cells, including CHO-S and DG44 cells;PER.C6® cells (Crucell); and NSO cells. In some embodiments, anti-CSF1Rheavy chains and/or anti-CSF1R light chains may be expressed in yeast.See, e.g., U.S. Publication No. US 2006/0270045 A1. In some embodiments,a particular eukaryotic host cell is selected based on its ability tomake desired post-translational modifications to the anti-CSF1R heavychains and/or anti-CSF1R light chains. For example, in some embodiments,CHO cells produce polypeptides that have a higher level of sialylationthan the same polypeptide produced in 293 cells.

Introduction of one or more nucleic acids into a desired host cell maybe accomplished by any method, including but not limited to, calciumphosphate transfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,etc. Nonlimiting exemplary methods are described, e.g., in Sambrook etal., Molecular Cloning, A Laboratory Manual, 3^(rd) ed. Cold SpringHarbor Laboratory Press (2001). Nucleic acids may be transiently orstably transfected in the desired host cells, according to any suitablemethod.

In some embodiments, one or more polypeptides may be produced in vivo inan animal that has been engineered or transfected with one or morenucleic acid molecules encoding the polypeptides, according to anysuitable method.

Purification of Anti-CSF1R Antibodies

Anti-CSF1R antibodies may be purified by any suitable method. Suchmethods include, but are not limited to, the use of affinity matrices orhydrophobic interaction chromatography. Suitable affinity ligandsinclude the CSF1R ECD and ligands that bind antibody constant regions.For example, a Protein A, Protein G, Protein A/G, or an antibodyaffinity column may be used to bind the constant region and to purify ananti-CSF1R antibody. Hydrophobic interactive chromatography, forexample, a butyl or phenyl column, may also suitable for purifying somepolypeptides. Many methods of purifying polypeptides are known in theart.

Cell-free Production of Anti-CSF1R Antibodies

In some embodiments, an anti-CSF1R antibody is produced in a cell-freesystem. Nonlimiting exemplary cell-free systems are described, e.g., inSitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, TrendsBiotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713(2003).

Methods of Detecting Factors

The present disclosure relates to methods of reducing one or morefactors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7,CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9. In some embodiments, asubject has an elevated level of one or more factors selected from IL-6,IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7,MMP-2, and MMP-9 prior to treatment with the antibodies describedherein. In some embodiments, the level of a factor is determined bydetecting the level of the protein. Nonlimiting exemplary amino acidsequences for human IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7, CXCL5,CXCL9, CXCL6, MMP-7, MMP-9, and MMP-2 are shown in SEQ ID NOs: 96 to108, respectively. Any native forms of the proteins, includingnaturally-occurring variants, such as variants comprising substitutionsand/or deletions (such as truncations), variants comprisingpost-translational modifications, splice variants, and allelic variants,are specifically contemplated.

In some embodiments, the level of the factor is determined by detectingthe level of the mRNA. Exemplary nucleotide sequences for human IL-6,IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7,MMP-2, and MMP-9 mRNA (or its complement, cDNA) are known in the art. Insome instances, the level of an mRNA may correlate with the level of theencoded protein, so that detection of the mRNA level may be used todetermine whether the level of the protein is, for example, elevatedprior to treatment, or has been reduced following treatment. In someembodiments, the level of the protein is determined. It should be noted,however, that it is not necessary to determine the level of thefactor(s) before or after treatment with the antibody in order to carryout the methods described herein. It can be assumed, in some instances,that a particular condition involves an elevated level of one or more ofthe recited factors, and therefore that a subject with the conditionwould benefit from a treatment that reduces one or more of the recitedfactors. Therefore, unless explicitly stated, detecting the level of oneor more factors before or after treatment is not required in order tocarry out the claimed methods.

Any method of detecting the level of a protein in a sample iscontemplated. One skilled in the art can select a suitable methoddepending on the type of sample being analyzed and the identity andnumber of proteins being detected. Nonlimiting exemplary such methodsinclude immunohistochemistry, ELISA, Western blotting, multiplex analytedetection (using, for example, Luminex technology), mass spectrometry,etc.

Similarly, any method of detecting the level of an mRNA in a sample iscontemplated. One skilled in the art can select a suitable methoddepending on the type of sample being analyzed and the identity andnumber of mRNAs being detected. Nonlimiting exemplary such methodsinclude RT-PCR, quantitative RT-PCR and microarray-based methods, etc.

Any method of determining the levels of CD16+ and/or CD16− monocytes iscontemplated. One skilled in the art can select a suitable methoddepending on the type of sample being analyzed. Nonlimiting exemplarymethods of determining the levels of CD16+ and/or CD16− monocytesinclude methods provided by commercial kits, such as CD16+ MoncyteIsolation Kit (Miltenyl Biotec, Bergisch Gladbach, Germany).

Therapeutic Compositions and Methods

Methods of Treating Diseases using Anti-CSF1R Antibodies

Provided herein are methods of reducing the level of at least one, atleast two, at least three, or at least four, at least five, at leastsix, at least seven, at least eight, at least nine, or at least tenfactors selected from factors selected from IL-6, IL-1β, IL-8, CCL2,CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9 in asubject comprising administering an antibody that binds CSF1R and blocksCSF1 and IL-34 ligand binding. In some embodiments, the method comprisesreducing at least one, at least two, at least three, or four factorsselected from IL-6, IL-1β, TNF-α, and CXCL10.

The amino acid sequences for exemplary mature human IL-6, IL-1β, IL-8,CCL2, CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-9, and MMP-2are shown in Table 8 (Table of Sequences; SEQ ID NOs: 96 to 108,respectively). Additional native mature sequences may also exist. Insome embodiments, native mature sequences have 1 to 10 or more aminoacids deleted from the amino terminus of the mature sequences shown inTable 8. In some embodiments, native mature sequences have one or moreamino acid additions, deletions, and/or substitutions in relative to themature sequences shown in Table 8. All of the native mature forms ofeach factor are intended to be encompassed herein.

Provided herein are methods of treating conditions associated withelevated levels of one or more factors selected from IL-6, IL-1β, IL-8,CCL2, CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9in a subject comprising administering an antibody that binds CSF1R andblocks CSF1 and IL-34 ligand binding. Exemplary conditions that areassociated with elevated levels of one or more of those factors include,but are not limited to, rheumatoid arthritis, juvenile idiopathicarthritis, Castleman's disease, psoriasis, psoriatic arthritis,ankylosing spondylitis, Crohn's disease, ulcerative colitis, lupuserythematosus, and inflammatory bowel disease. In some embodiments, theantibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding isselected from huAb1 to huAb16, described herein. In some embodiments,the antibody is huAb1.

In some embodiments, a method of reducing the level of IL-6 in a subjectis provided, comprising administering to the subject an antibody thatbinds CSF1R and blocks CSF1 and IL-34 ligand binding. Reducing the levelof IL-6 is beneficial, in some embodiments, in the treatment of acondition associated with elevated IL-6, such as rheumatoid arthritis,juvenile idiopathic arthritis, and Castleman's disease. In someembodiments, a method of reducing the level of TNF-α in a subject isprovided, comprising administering to the subject an antibody that bindsCSF1R and blocks CSF1 and IL-34 ligand binding. Reducing the level ofTNF-α is beneficial, in some embodiments, in the treatment of acondition associated with elevated TNF-α, such as rheumatoid arthritis,juvenile idiopathic arthritis, psoriasis, psoriatic arthritis,ankylosing spondylitis, Crohn's disease, and ulcerative colitis. In someembodiments, a method of reducing the level of IL-1β in a subject isprovided, comprising administering to the subject an antibody that bindsCSF1R and blocks CSF1 and IL-34 ligand binding. Reducing the level ofIL-1β is beneficial, in some embodiments, in the treatment of acondition associated with elevated IL-1β, such as rheumatoid arthritisand juvenile idiopathic arthritis. In any of the embodiments herein, theantibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding maybe selected from huAb1 to huAb16, described herein. In any of theembodiments herein, the antibody may be huAb1.

In some embodiments, a method comprises reducing IL-6 and IL-1β. In someembodiments, the method comprises reducing IL-6 and TNF-α. In someembodiments, a method comprises reducing IL-6 and CXCL10. In someembodiments, a method comprises reducing IL-1β and TNF-α. In someembodiments, a method comprises reducing IL-1β and CXCL10. In someembodiments, a method comprises reducing TNF-α and CXCL10. In someembodiments, a method comprises reducing IL-6, IL-1β, and TNF-α. In someembodiments, a method comprises reducing IL-6, IL-1β, and CXCL10. Insome embodiments, a method comprises reducing IL-6, TNF-α, and CXCL10.In some embodiments, a method comprises reducing TNF-α, IL-1β, andCXCL10. In some embodiments, a method comprises reducing IL-6, IL-1β,TNF-α, and CXCL10.

Methods of treating an inflammatory condition are provided, comprisingadministering to a subject with an inflammatory condition an effectiveamount of an antibody that binds CSF1R and blocks CSF1 and IL-34 ligandbinding. In some embodiments, a method of treating an inflammatorycondition comprises reducing the level of one or more factors selectedfrom IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6,MMP-7, MMP-2, and MMP-9 in a subject with an inflammatory condition,comprising administering an antibody that binds CSF1R and blocks CSF1and IL-34 ligand binding. In some embodiments, the method comprisesreducing at least one, at least two, at least three, or four factorsselected from IL-6, IL-1β, TNF-α, and CXCL10. In some embodiments, theantibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding isselected from huAb1 to huAb16, described herein. In some embodiments,the antibody is huAb1. Nonlimiting exemplary inflammatory conditionsinclude rheumatoid arthritis, juvenile idiopathic arthritis, Castleman'sdisease, psoriasis, psoriatic arthritis, ankylosing spondylitis, Crohn'sdisease, ulcerative colitis, lupus erythematosus, inflammatory boweldisease, inflammatory arthritis, and CD16+ disorders.

Methods of treating inflammatory arthritis are provided, comprisingadministering to a subject with an inflammatory arthritis an effectiveamount of an antibody that binds CSF1R and blocks CSF1 and IL-34 ligandbinding. In some embodiments, a method of treating inflammatoryarthritis comprises reducing the level of one or more factors selectedfrom IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6,MMP-7, MMP-2, and MMP-9 in a subject with inflammatory arthritis,comprising administering an effective amount of an antibody that bindsCSF1R and blocks CSF1 and IL-34 ligand binding. In some embodiments, themethod comprises reducing at least one, at least two, at least three, orfour factors selected from IL-6, IL-1β, TNF-α, and CXCL10. In someembodiments, the antibody that binds CSF1R and blocks CSF1 and IL-34ligand binding is selected from huAb1 to huAb16, described herein. Insome embodiments, the antibody is huAb1.

In some embodiments, in addition to reducing the level of one or morefactors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7,CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9, administering to a subjectwith inflammatory arthritis an effective amount of an antibody thatbinds CSF1R and blocks CSF1 and IL-34 ligand binding, such as huAb1 tohuAb16, reduces inflammation, reduces pannus formation, reducescartilage damage, reduces bone resorption, reduces macrophage numbers inthe joints, reduces autoantibody formation, and/or reduces bone loss.

In some embodiments, in addition to reducing the level of one or morefactors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7,CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9, administering to a subjectwith inflammatory arthritis an effective amount of an antibody thatbinds CSF1R and blocks CSF1 and IL-34 ligand binding, such as huAb1 tohuAb16, reduces inflammation. Reducing inflammation, in someembodiments, comprises reducing erythrocyte sedimentation rate and/orreducing the levels of C-reactive proteins in blood. When inflammationis present in a subject, the erythrocyte sedimentation rate increases,possibly due to increased levels of fibrinogen in the blood. Theerythrocyte sedimentation rate may be determined by any method in theart, including, but not limited to, calculating the rate by measuringthe change in height of anticoagulated erythrocytes in one hour in aWestergren tube. See also Procedures for the Erythrocyte SedimentationRate Test; Approved Standard—Fifth Edition. CLSI document H02-A5. Wayne,Pa.: Clinical and Laboratory Standards Institute; 2011. Levels ofC-reactive protein in blood may be determined by any methods in the art,including but not limited to using the RAPITEX® CRP test kit (Siemens).

Reducing inflammation, in some embodiments, comprises reducingperipheral edema, which is tissue swelling due to the buildup of fluids.Peripheral edema may occur, in some instances, in the ankles, feet,legs, and/or calves of a subject with rheumatoid arthritis. Reducinginflammation, in some embodiments, comprises reducing infiltration ofinflammatory cells in the synovium of one or more affected joints.Synovial fluid may be collected, in some embodiments, by athrocentesis.

In some embodiments, in addition to reducing the level of one or morefactors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7,CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9, administering to a subjectwith inflammatory arthritis an effective amount of an antibody thatbinds CSF1R and blocks CSF1 and IL-34 ligand binding, such as huAb1 tohuAb16, reduces pannus formation. Reducing pannus formation, in someembodiments, comprises reducing infiltration of pannus into cartilageand/or subchondrial bone, and/or reducing hard tissue destructionresulting from pannus infiltration. Pannus formation can be measured byany method in the art, including, but not limited to, imaging one ormore affected joints. Nonlimiting exemplary imaging techniques fordetecting pannus formation include magnetic resonance imaging (MRI),computed tomography (CT) scan, arthroscopy, ultrasonography, duplexultrasonography, and power doppler imaging. In some embodiments, theprogression of pannus formation is slowed following administration ofthe antibody and/or during a particular time interval during which thesubject is undergoing treatment with the antibody. The treatment may bea single dose or multiple doses.

In some embodiments, in addition to reducing the level of one or morefactors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7,CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9, administering to a subjectwith inflammatory arthritis an effective amount of an antibody thatbinds CSF1R and blocks CSF1 and IL-34 ligand binding, such as huAb1 tohuAb16, reduces cartilage damage. Reducing cartilage damage, in someembodiments, comprises reducing chondrocyte loss, reducing collagendisruption, and/or reducing cartilage loss. Cartilage damage can bemeasured by any method in the art, including, but not limited to,imaging one or more affected joints. Nonlimiting exemplary imagingtechniques for detecting cartilage damage include MRI, CT scan,arthroscopy, and x-ray imaging. In some embodiments, the progression ofcartilage damage is slowed following administration of the antibodyand/or during a particular time interval during which the subject isundergoing treatment with the antibody. The treatment may be a singledose or multiple doses.

In some embodiments, in addition to reducing the level of one or morefactors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7,CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9, administering to a subjectwith inflammatory arthritis an effective amount of an antibody thatbinds CSF1R and blocks CSF1 and IL-34 ligand binding, such as huAb1 tohuAb16, reduces bone resorption. Reducing bone resorption, in someembodiments, comprises reducing the number of osteoclasts in jointsaffected by rheumatoid arthritis.

In some embodiments, bone resorption may be measured by determining thelevel of TRAPSb in plasma from the subject, wherein an elevated level ofTRAPSb indicates elevated bone resorption in the subject. Thus, in someembodiments, a reduced level of TRAPSb indicates a reduction in boneresorption. TRAPSb levels may be determined, in certain instances,before and after treatment with an antibody that binds CSF1R, and/or maybe determined periodically throughout the course of treatment to monitorthe effectiveness of the treatment in reducing bone loss. TRAPSb levelsmay be determined using any method in the art, including, but notlimited to, ELISA (including FAICEA, or fragments absorbed immunocaptureenzymatic assay; see, e.g., Quidel® TRAPSb assay, TECOmedical Group,Sissach, Switzerland).

In some embodiments, in addition to reducing the level of one or morefactors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7,CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9, administering to a subjectwith inflammatory arthritis an effective amount of an antibody thatbinds CSF1R and blocks CSF1 and IL-34 ligand binding, such as huAb1 tohuAb16, reduces bone loss. Bone loss may be determined using any methodin the art, including, but not limited to, x-ray imaging, MRI, CT, bonedensitometry, single and dual photon absorptiometry (SPA, DPA), singleand dual energy x-ray absorptiometry (SXA, DXA), ultrasonography,scintigraphy, and by measuring levels of serum markers of bone formationand resorption. Nonlimiting exemplary serum markers of bone formationand bone resorption are shown in Table 2.

TABLE 2 Serum markers of bone formation and resorption Formation MarkersResorption Markers Serum osteocalcin (OC) Serum and urinaryhydroxyproline (Hyp) Serum total alkaline Urinary total pyridinoline(Pyr) phosphatase (ALP) Serum bone specific alkaline Urinary totaldeoxypyridinoline phosphatase (BSAP, BALP, (dPyr) or B-ALP) Serumprocollagen I Urinary free pyridinoline carboxyterminal (f-Pyr, alsoknown as Pyrilinks ® propeptide (PICP) (Metra Biosystems)) Serumprocollagen type 1 Urinary free deoxypyridinoline N-terminal propeptide(f-dPyr, also known as Pyrilinks-D ®) (PINP) Bone sialoprotein Serum andurinary collagen type I cross-linked N-telopeptide (NTx, also referredto as Osteomark) Serum and urinary collagen type I cross-linked C-terminal telopeptide (CTx, also referred to as CrossLaps ®) Serumcarboxyterminal telopeptide of type I collagen (ITCP) Tartrate-resistantacid phosphatase (TRAP or TRACP)

In some embodiments, the progression of bone loss is slowed followingadministration of the antibody and/or during a particular time intervalduring which the subject is undergoing treatment with the antibody. Thetreatment may be a single dose or multiple doses.

In some embodiments, in addition to reducing the level of one or morefactors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7,CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9, administering to a subjectwith inflammatory arthritis an effective amount of an antibody thatbinds CSF1R and blocks CSF1 and IL-34 ligand binding, such as huAb1 tohuAb16, reduces autoantibody levels. The levels of autoantibodies may bedetermined by any method in the art. In some embodiments, autoantibodylevels are determined by the level of rheumatoid factor (RF) and/oranti-citrullinated protein antibodies (ACPA) and/or anti-nuclearantibodies (ANA).

In some embodiments, in addition to reducing the level of one or morefactors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7,CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9, administering to a subjectwith inflammatory arthritis an effective amount of an antibody thatbinds CSF1R and blocks CSF1 and IL-34 ligand binding, such as huAb1 tohuAb16, substantially reduces the number of monocyte lineage cells, suchas macrophages and/or CD16+ monocytes, in joints (including synovialfluid) affected by the inflammatory arthritis.

In some embodiments, in addition to reducing the level of one or morefactors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7,CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9, administering to a subjectwith inflammatory arthritis an effective amount of an antibody thatbinds CSF1R and blocks CSF1 and IL-34 ligand binding, such as huAb1 tohuAb16, substantially reduces the number of CD16+ monocytes. In someembodiments, the subject has an autoimmune condition selected fromrheumatoid arthritis and SLE (lupus). In some embodiments, followingadministration of an effective amount of an antibody that binds CSF1Rand blocks CSF1 and IL-34 ligand binding, the number of CD16− monocytesis substantially unchanged. In some embodiments, CD16+ monocytes arereduced to a greater extent than CD16− monocytes are reduced when anantibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding isadministered to the subject. In some embodiments, CD16+ monocytes arereduced by at least 20%, at least 30%, at least 50%, at least 60%, atleast 70%, at least 80%, or at least 90%. In some embodiments, CD16−monocytes are reduced by less than 30%, less than 20%, or less than 10%.In some embodiments, the CD16+ monocytes are CD16+ peripheral bloodmonocytes. In some embodiments, the CD16− monocytes are CD16− peripheralblood monocytes.

In some embodiments, methods of treating a CD16+ disorder are provided,comprising administering to a subject with a CD16+ disorder an effectiveamount of an antibody that binds CSF1R and blocks CSF1 and IL-34 ligandbinding, such as huAb1 to huAb16, wherein the antibody reduces the levelof at least one, at least two, at least three, or at least four, atleast five, at least six, at least seven, at least eight, at least nine,or at least ten factors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10,TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9. Nonlimitingexemplary CD16+ disorders include rheumatoid arthritis, juvenileidiopathic arthritis, Castleman's disease, psoriasis, psoriaticarthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis,lupus erythematosus, and inflammatory bowel disease. In someembodiments, a subject with a CD16+ disorder has an elevated level ofCD16+ monocytes, compared to the level of CD16+ monocytes in a healthyindividual or a pool of healthy individuals. In some embodiments, asubject with a CD16+ disorder has an elevated level of CD16+ monocytes,compared to the subject's CD16+ monocyte level prior to developing theCD16+ disorder (for example, in some embodiments, substantially prior todeveloping any symptoms of the CD16+ disorder such that the subjectwould, in retrospect, be considered “healthy” at the time).

In some embodiments, methods of identifying subjects who may benefitfrom an antibody that binds CSF1R, wherein the antibody blocks bindingof CSF1 to CSF1R and blocks binding of IL-34 to CSF1R (such as huAb1 tohuAb16) are provided. In some such embodiments, a method comprisesdetermining the level of at least one, at least two, at least three, orat least four, at least five, at least six, at least seven, at leasteight, at least nine, or at least ten factors selected from IL-6, IL-1β,IL-8, CCL2, CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, andMMP-9 in the subject. In some embodiments, an elevated level of at leastone of the factors in the subject indicates that the subject may benefitfrom the antibody that binds CSF1R. In some embodiments, the subject hasa CD16+ disorder. In some embodiments, the subject has rheumatoidarthritis. In some embodiments, the subject has an elevated level ofCD16+ monocytes.

In some embodiments, methods of predicting responsiveness in a subjectsuffering from an inflammatory condition to an antibody that bindsCSF1R, wherein the antibody blocks binding of CSF1 to CSF1R and blocksbinding of IL-34 to CSF1R (such as huAb1 to huAb16) are provided. Insome such embodiments, a method comprises determining the level of atleast one, at least two, at least three, or at least four, at leastfive, at least six, at least seven, at least eight, at least nine, or atleast ten factors selected from IL-6, IL-1β, IL-8, CCL2, CXCL10, TNF-α,CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9 in the subject. Insome embodiments, an elevated level of at least one of the factors inthe subject indicates that the subject is more likely to respond to theantibody that binds CSF1R. In some embodiments, the subject has a CD16+disorder. In some embodiments, the subject has rheumatoid arthritis. Insome embodiments, the subject has an elevated level of CD16+ monocytes.

In some embodiments of the methods described herein, the subject has acondition that is resistant to methotrexate (e.g., the subject ismethotrexate inadequate responder). A subject with a condition that isresistant to methotrexate, such as a subject who is a methotrexateinadequate responder, may have previously responded to methotrexate, butmay have become resistant to methotrexate, or the subject may have neverresponded to methotrexate. Resistance to methotrexate means that aspectsof the condition that would be expected to improve following a standarddose of methotrexate do not improve, and/or improvement only occurs ifgreater than a standard dose of methotrexate is administered. In someembodiments, a methotrexate inadequate responder has experienced, or isexperiencing, an inadequate response to methotrexate after receiving astandard dose for at least two weeks, at least three weeks, at leastfour weeks, at least six weeks, or at least twelve weeks. A “standard”dose is determined by a medical professional, and may depend on thesubject's age, weight, healthy history, severity of disease, thefrequency of dosing, etc.

In some embodiments of the methods described herein, the subject is aTNF inhibitor inadequate responder. A subject who is a TNF inhibitorinadequate responder, may have previously responded to a TNF inhibitor,but may have become less responsive to the TNF inhibitor, or the subjectmay have never responded to the TNF inhibitor. Inadequate response to aTNF inhibitor means that aspects of the condition that would be expectedto improve following a standard dose of the TNF inhibitor do notimprove, and/or improvement only occurs if greater than a standard doseis administered. In some embodiments, a TNF inhibitor inadequateresponder has experienced, or is experiencing, an inadequate response tothe TNF inhibitor after receiving a standard dose for at least twoweeks, at least three weeks, at least four weeks, at least six weeks, orat least twelve weeks. A “standard” dose is determined by a medicalprofessional, and may depend on the subject's age, weight, healthyhistory, severity of disease, the frequency of dosing, etc. In someembodiments, a TNF inhibitor inadequate responder has experienced, or isexperiencing, an inadequate response to a TNF inhibitor selected frominfliximab, adalimumab, certolizumab pegol, golimumab, and etanercept.

In some embodiments, methods of treating a methotrexate inadequateresponder are provided. In some embodiments, a method comprisesadministering to the methotrexate inadequate responder an antibody thatbinds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R andblocks binding of IL-34 to CSF1R, such as huAb1 to huAb16. In someembodiments, the inadequate responder has a CD16+ disorder. In someembodiments, the CD16+ disorder is selected from rheumatoid arthritis,juvenile idiopathic arthritis, Castleman's disease, psoriasis, psoriaticarthritis, ankylosing spondylitis, Crohn's disease, and ulcerativecolitis, lupus erythematosus, and inflammatory bowel disease. In someembodiments, the CD16+ disorder is rheumatoid arthritis. In someembodiments, the antibody substantially reduces the number of CD16+monocytes. In some embodiments, the number of CD16− monocytes aresubstantially unchanged following administration of the antibody. Insome embodiments, the level of at least one, at least two, at leastthree, or at least four, at least five, at least six, at least seven, atleast eight, at least nine, or at least ten factors selected from IL-6,IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7,MMP-2, and MMP-9 in the inadequate responder is reduced followingadministration of the antibody. In some embodiments, the inadequateresponder has an elevated level of CD16+ monocytes, for example, ascompared to the level of CD16+monocytes in a healthy individual or poolof healthy individuals. In some embodiments, the antibody substantiallyreduces the number of CD16+ monocytes. In some embodiments, the numberof CD16− monocytes are substantially unchanged following administrationof the antibody.

In some embodiments, methods of treating a TNF inhibitor inadequateresponder are provided. In some embodiments, a method comprisesadministering to the TNF inhibitor inadequate responder an antibody thatbinds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R andblocks binding of IL-34 to CSF1R, such as huAb1 to huAb16. In someembodiments, the inadequate responder has a CD16+ disorder. In someembodiments, the CD16+ disorder is selected from rheumatoid arthritis,juvenile idiopathic arthritis, Castleman's disease, psoriasis, psoriaticarthritis, ankylosing spondylitis, Crohn's disease, and ulcerativecolitis, lupus erythematosus, and inflammatory bowel disease. In someembodiments, the CD16+ disorder is rheumatoid arthritis. In someembodiments, the antibody substantially reduces the number of CD16+monocytes. In some embodiments, the number of CD16− monocytes aresubstantially unchanged following administration of the antibody. Insome embodiments, the level of at least one, at least two, at leastthree, or at least four, at least five, at least six, at least seven, atleast eight, at least nine, or at least ten factors selected from IL-6,IL-1β, IL-8, CCL2, CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7,MMP-2, and MMP-9 in the inadequate responder is reduced followingadministration of the antibody. In some embodiments, the inadequateresponder has an elevated level of CD16+ monocytes, for example, ascompared to the level of CD16+ monocytes in a healthy individual or poolof healthy individuals. In some embodiments, the antibody substantiallyreduces the number of CD16+ monocytes. In some embodiments, the numberof CD16− monocytes are substantially unchanged following administrationof the antibody.

Routes of Administration and Carriers

In various embodiments, anti-CSF1R antibodies may be administered invivo by various routes, including, but not limited to, oral,intra-arterial, parenteral, intranasal, intramuscular, intracardiac,intraventricular, intratracheal, buccal, rectal, intraperitoneal,intradermal, topical, transdermal, and intrathecal, or otherwise byimplantation or inhalation. The subject compositions may be formulatedinto preparations in solid, semi-solid, liquid, or gaseous forms;including, but not limited to, tablets, capsules, powders, granules,ointments, solutions, suppositories, enemas, injections, inhalants, andaerosols. A nucleic acid molecule encoding an anti-CSF1R antibody may becoated onto gold microparticles and delivered intradermally by aparticle bombardment device, or “gene gun,” as described in theliterature (see, e.g., Tang et al., Nature 356:152-154 (1992)). Theappropriate formulation and route of administration may be selectedaccording to the intended application.

In various embodiments, compositions comprising anti-CSF1R antibodiesare provided in formulations with a wide variety of pharmaceuticallyacceptable carriers (see, e.g., Gennaro, Remington: The Science andPractice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20^(th)ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug DeliverySystems, 7^(th) ed., Lippencott Williams and Wilkins (2004); Kibbe etal., Handbook of Pharmaceutical Excipients, 3rd ed., PharmaceuticalPress (2000)). Various pharmaceutically acceptable carriers, whichinclude vehicles, adjuvants, and diluents, are available. Moreover,various pharmaceutically acceptable auxiliary substances, such as Phadjusting and buffering agents, tonicity adjusting agents, stabilizers,wetting agents and the like, are also available. Non-limiting exemplarycarriers include saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof.

In various embodiments, compositions comprising anti-CSF1R antibodiesmay be formulated for injection, including subcutaneous administration,by dissolving, suspending, or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids, or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives. In various embodiments, the compositions may beformulated for inhalation, for example, using pressurized acceptablepropellants such as dichlorodifluoromethane, propane, nitrogen, and thelike. The compositions may also be formulated, in various embodiments,into sustained release microcapsules, such as with biodegradable ornon-biodegradable polymers. A non-limiting exemplary biodegradableformulation includes poly lactic acid-glycolic acid polymer. Anon-limiting exemplary non-biodegradable formulation includes apolyglycerin fatty acid ester. Certain methods of making suchformulations are described, for example, in EP 1 125 584 A1.

Pharmaceutical packs and kits comprising one or more containers, eachcontaining one or more doses of an anti-CSF1R antibody are alsoprovided. In some embodiments, a unit dosage is provided wherein theunit dosage contains a predetermined amount of a composition comprisingan anti-CSF1R antibody, with or without one or more additional agents.In some embodiments, such a unit dosage is supplied in single-useprefilled syringe for injection. In various embodiments, the compositioncontained in the unit dosage may comprise saline, sucrose, or the like;a buffer, such as phosphate, or the like; and/or be formulated within astable and effective Ph range. Alternatively, in some embodiments, thecomposition may be provided as a lyophilized powder that may bereconstituted upon addition of an appropriate liquid, for example,sterile water. In some embodiments, the composition comprises one ormore substances that inhibit protein aggregation, including, but notlimited to, sucrose and arginine. In some embodiments, a composition ofthe invention comprises heparin and/or a proteoglycan.

Pharmaceutical compositions are administered in an amount effective fortreatment or prophylaxis of the specific indication. The therapeuticallyeffective amount is typically dependent on the weight of the subjectbeing treated, his or her physical or health condition, theextensiveness of the condition to be treated, or the age of the subjectbeing treated. In general, anti-CSF1R antibodies may be administered inan amount in the range of about 10 μg/kg body weight to about 100 mg/kgbody weight per dose. In some embodiments, anti-CSF1R antibodies may beadministered in an amount in the range of about 50 μg/kg body weight toabout 5 mg/kg body weight per dose. In some embodiments, anti-CSF1Rantibodies may be administered in an amount in the range of about 100μg/kg body weight to about 10 mg/kg body weight per dose. In someembodiments, anti-CSF1R antibodies may be administered in an amount inthe range of about 100 μg/kg body weight to about 20 mg/kg body weightper dose. In some embodiments, anti-CSF1R antibodies may be administeredin an amount in the range of about 0.5 mg/kg body weight to about 20mg/kg body weight per dose.

The anti-CSF1R antibody compositions may be administered as needed tosubjects. Determination of the frequency of administration may be madeby persons skilled in the art, such as an attending physician based onconsiderations of the condition being treated, age of the subject beingtreated, severity of the condition being treated, general state ofhealth of the subject being treated and the like. In some embodiments,an effective dose of an anti-CSF1R antibody is administered to a subjectone or more times. In various embodiments, an effective dose of ananti-CSF1R antibody is administered to the subject once a month, lessthan once a month, such as, for example, every two months or every threemonths. In other embodiments, an effective dose of an anti-CSF1Rantibody is administered more than once a month, such as, for example,every three weeks, every two weeks or every week. An effective dose ofan anti-CSF1R antibody is administered to the subject at least once. Insome embodiments, the effective dose of an anti-CSF1R antibody may beadministered multiple times, including for periods of at least a month,at least six months, or at least a year.

Combination Therapy

Anti-CSF1R antibodies may be administered alone or with other modes oftreatment. They may be provided before, substantially contemporaneouswith, or after other modes of treatment, for example, surgery,chemotherapy, radiation therapy, or the administration of a biologic,such as another therapeutic antibody. For treatment of inflammatoryarthritis (including rheumatoid arthritis, juvenile idiopathicarthritis, ankylosing spondylitis, etc.), anti-CSF1R antibodies may beadministered with other therapeutic agents, for example, methotrexate,anti-TNF agents, including anti-TNF antibodies such as Remicade®(infliximab), Humira® (adalimumab), Simponi® (golimumab), andcertolizumab pegol, and soluble TNF receptors, such as Enbrel®(etanercept); glucocorticoids such as prednisone; leflunomide;azathioprine; JAK inhibitors such as CP 590690; SYK inhibitors such asR788; anti-IL-6 agents, including anti-IL-6 antibodies such aselsilimomab, siltuximab, and sirukumab, and anti-IL-6R antibodies suchas Actermra® (tocilizumab); anti-CD-20 agents, including anti-CD20antibodies such as Rituxin® (rituximab), ibritumomab tiuxetan,ofatumumab, ocrelizumab, veltuzumab, and tositumomab; anti-CD19 agents,such as anti-CD19 antibodies; anti-GM-CSF agents, such as anti-GM-CSFantibodies and anti-GM-CSFR antibodies; anti-IL-1 agents, such as IL-1receptor antagonists, including anakinra; CTLA-4 agonists, such asCTLA4-Ig fusions, including abatacept and belatacept; immunosuppressantssuch as cyclosporine.

For treatment of systemic lupus erythematosus, anti-CSF1R antibodies maybe administered with other therapeutic agents, for example,hydroxychloroquine (Plaquenil®); corticosteroids, such as prednisone,methylprednisone, and prednisolone; immunosuppressants, such ascyclophosphamide (Cytoxan®), azathioprine (Imuran®, Azasan®),mycophenolate (Cellcept®), leflunomide (Arava®), methotrexate(Trexall™), and belimumab (Benlysta®).

For treatment of multiple sclerosis, anti-CSF1R antibodies may beadministered with other therapeutic agents, for example, interferonalpha; interferon beta; prednisone; anti-alpha4 integrin antibodies suchas Tysabri®; anti-CD20 antibodies such as Rituxan®; FTY720 (fingolimod;Gilenya®); and cladribine (Leustatin®).

EXAMPLES

The examples discussed below are intended to be purely exemplary of theinvention and should not be considered to limit the invention in anyway. The examples are not intended to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (for example, amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Example 1: Humanized Anti-CSF1R Antibodies

Various humanized anti-CSF1R antibodies were developed previously. See,e.g., PCT Publication No. WO 2011/140249.

The sequences for each of the humanized heavy chain variable regions andhumanized light chain variable regions, aligned with the sequences ofthe parental chimeric antibody variable regions and the sequences of thehuman acceptor variable framework regions are shown in FIGS. 1A-C (heavychains) and 2 (light chains). The changes in humanized variable regionsequences relative to the human acceptor variable framework regionsequences are boxed. Each of the CDRs for each of the variable regionsis shown in a boxed region, and labeled as “CDR” above the boxedsequences.

Table 8, below, shows the full sequences for the humanized heavy chainsand humanized light chains of antibodies huAb1 to huAb16. The name andSEQ ID Nos of the humanized heavy chain and humanized light chain ofeach of those antibodies is shown in Table 3.

TABLE 3 Humanized heavy chains and light chains of huAb1 to huAb16Humanized Humanized SEQ Humanized SEQ antibody HC ID NO LC ID NO huAb1h0301-H0 53 h0301-L0 60 huAb2 h0301-H1 54 h0301-L0 60 huAb3 h0301-H2 55h0301-L0 60 huAb4 h0301-H0 53 h0301-L1 61 huAb5 h0301-H1 54 h0301-L1 61huAb6 h0301-H2 55 h0301-L1 61 huAb7 h0302-H1 56 h0302-L0 62 huAb8h0302-H1 56 h0302-L1 63 huAb9 h0302-H1 56 h0302-L2 64 huAb10 h0302-H2 57h0302-L0 62 huAb11 h0302-H2 57 h0302-L1 63 huAb12 h0302-H2 57 h0302-L264 huAb13 h0311-H1 58 h0311-L0 65 huAb14 h0311-H1 58 h0311-L1 66 huAb15h0311-H2 59 h0311-L0 65 huAb16 h0311-H2 59 h0311-L1 66

The 16 humanized antibodies were tested for binding to human, cynomolgusmonkey, and mouse CSF1R ECD, as described previously. See, e.g., PCTPublication No. WO 2011/140249. The antibodies were found to bind toboth human and cynomolgus monkey CSF1R ECD, but not to mouse CSF1R ECD.The humanized antibodies were also found to block binding of CSF1 andIL-34 to both human and mouse CSF1R and to inhibit CSF1-induced andIL-34-induced phosphorylation of human CSF1R expressed in CHO cells.See, e.g., PCT Publication No. WO 2011/140249.

The k_(a), k_(d), and K_(D) for binding to human CSF1R ECD werepreviously determined and are shown in Table 4. See, e.g., PCTPublication No. WO 2011/140249.

TABLE 4 Humanized antibody binding affinity for human CSF1R huAb k_(a)(M⁻¹s⁻¹) K_(d) (s⁻¹) K_(D) (Nm) huAb 0301-L0H0 3.22 × 10⁶ 1.11 × 10⁻⁰³0.35 huAb 0301-L0H1 3.56 × 10⁶ 1.22 × 10⁻⁰³ 0.34 huAb 0301-L0H2 2.32 ×10⁶ 6.60 × 10⁻⁰⁴ 0.28 huAb 0301-L1H0 3.29 × 10⁶ 1.15 × 10⁻⁰³ 0.35 huAb0301-L1H1 2.87 × 10⁶ 9.21 × 10⁻⁰⁴ 0.32 huAb 0301-L1H2 2.95 × 10⁶ 7.42 ×10⁻⁰⁴ 0.25 huAb 0302-L0H1 3.54 × 10⁶ 3.69 × 10⁻⁰³ 1.04 huAb 0302-L1H13.47 × 10⁶ 4.04 × 10⁻⁰³ 1.17 huAb 0302-L2H1 1.60 × 10⁶ 9.14 × 10⁻⁰⁴ 0.57huAb 0302-L0H2 3.40 × 10⁶ 1.79 × 10⁻⁰³ 0.53 huAb 0302-L1H2 2.71 × 10⁶1.53 × 10⁻⁰³ 0.56 huAb 0302-L2H2 1.84 × 10⁶ 8.40 × 10⁻⁰⁴ 0.46 huAb0311-L0H1 1.22 × 10⁶ 5.40 × 10⁻⁰⁴ 0.44 huAb 0311-L1H1 1.32 × 10⁶ 6.64 ×10⁻⁰⁴ 0.50 huAb 0311-L0H2 1.34 × 10⁶ 4.73 × 10⁻⁰⁴ 0.35 huAb 0311-L1H21.51 × 10⁶ 6.09 × 10⁻⁰⁴ 0.40

Example 2: HuAb1 Alters Cytokine and Certain Matrix MetalloproteinaseProduction in Synovial Biopsy Explants

Synovial tissue samples were obtained from the joints of rheumatoidarthritis patients. Patients had clinically active disease and tissuewas obtained from clinically active joints. All patients providedwritten informed consent and these studies were approved by the MedicalEthics Committee of the Academic Medical Center (AMC) at the Universityof Amsterdam. The clinical characteristics of the six patients from whombiopsy samples were taken are shown in Table 5.

TABLE 5 Clinical features of patients with RA (n = 6) Disease Ageduration ESR CRP Biopsy (y) Sex (y) (mm/h) (mg/l) DAS28 RF ACCP 1 56 M10 12 3.4 3.64 positive negative 2 42 F 21 1 2.59 positive positive 3 68F 23 10 2 4.01 positive positive 4 78 F 10 44 51.1 4.32 positivepositive 5 61 M 18 positive positive 6 71 F 25 14 3.3 4.21 negativenegative ACCP, anti-cyclic citrullinated peptide; CRP, C-reactivepeptide; DAS28, 28-joint disease activity score; ESR, erythrocytesedimentation rate; RF, rheumatoid factor.

Synovial biopsy samples with a volume of approximately 5 mm³ werecultured in triplicate in complete medium comprising DMEM (LifeTechnologies, Grand Island, N.Y.) with 2 Mm L-glutamine, 100 U/mlpenicillin, 50 mg/ml gentamicin, 20 Mm HEPES buffer, and 10% FCS.Cultures were performed at 37° C. in a 5% CO₂/95% air-humidifiedenvironment. Synovial samples were cultured for 4 days in the absence orpresence of increasing concentrations of huAb1 or control IgG4 antibodyET904 (Eureka Therapeutics, Emeryville, Calif.). Cell-free supernatantswere collected and stored at −80° C. in two separate aliquots. Onealiquot was evaluated for production of IL-6 by ELISA. The other aliquotwas evaluated for multiplex analysis of cytokine and matrixmetalloproteinase production using Luminex® technology (Millipore,Billerica, Mass.; Catalog Nos. MPXHCYTO-60K-08, MPXHCYP2-62K-02,MPXHCYP3-63K-04, HMMP2-55K-03). The three tissue fragments in eachculture condition were pooled, snap-frozen and preserved for Mrnaexpression analysis.

The results of the IL-6 production analysis by ELISA are shown in Table6.

TABLE 6 Effect of huAb1 treatment on intact synovial biopsy IL-6production (ELISA) IL-6 Levels (pg/ml) per mg of Tissue 0.1 1 10 0.1 110 μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml Biopsy Medium IgG4 IgG4 IgG4HuAb1 HuAb1 HuAb1 1  1.26  51.92  99.27  21.43  60.74  36.06  17.05 2151.05 151.21  127.24  7.81  31.50  13.19  4.62 3  77.76 428.04  292.85116.80 180.45  13.24  30.58 4  1.77  10.62  90.36 291.33  6.75   7.64 30.03 5 323.00 385.10 1285.00 470.90 528.50 1243.00 352.40 6 176.90111.40  99.02  62.54  33.46  38.59  41.92 Mean 121.96 189.72  332.29161.80 140.23  225.29  79.43 SEM  50.08  71.57 193.1  74.75  81.59 203.6 54.84

IL-6 production was reduced in all samples after culturing for 4 days inthe presence of 1 μg/ml or 10 μg/ml huAb1, as compared to incubation inthe same concentration of control antibody. IL-6 production was reducedin four of the six samples after culturing for 4 days in the presence of0.1 μg/ml huAb1, as compared to incubation in 0.1 μg/ml of controlantibody. FIG. 3 shows a plot of the reduction in IL-6 production in thefour samples after culturing for 4 days in the presence of 1 μg/mlcontrol antibody or 1 μg/ml huAb1. The mean decrease in IL-6 productionwas statistically significant at both 1 μg/ml and 10 μg/ml huAb1(p=0.0313 at each dose).

FIGS. 4A-L show the results of the multiplex analysis after culturingfour of the synovial biopsy explants in 1 μg/ml huAb1 or controlantibody for 4 days. Levels of IL-6, IL-1β, IL-8, CCL2 (also referred toas MCP-1), CXCL10, TNF-α, CCL7, CXCL5, CXCL9, CXCL6, and MMP-9 werereduced in all four samples after incubation with 1 μg/ml huAb1,relative to incubation with 1 μg/ml control antibody. Levels of MMP-7were reduced in the two samples with measurable levels of MMP-7 in thecontrol antibody-treated groups.

Table 7 below shows the results of the multiplex analysis for the foursynovial biopsy explants shown in FIGS. 4A-L. In Table 7, the averagecytokine level in the four explants is shown after incubation in mediumalone, 0.1 μg/ml, 1 μg/ml, or 10 μg/ml IgG4 control, or 0.1 μg/ml, 1μg/ml, or 10 μg/ml huAb1.

Multiplex analysis of MMP-2 and MMP-9 levels after culturing twoadditional synovial biopsy explants in the presence of medium alone, 0.1μg/ml, 1 μg/ml, or 10 μg/ml IgG4 control, or 0.1 μg/ml, 1 μg/ml, or 10μg/ml huAb1 was performed substantially as described above. Table 8below shows the results of the multiplex analysis for all six synovialbiopsy explants tested for MMP-2 and MMP-9 levels.

Multiplex analysis of MMP-7 levels after culturing one additionalsynovial biopsy explants in the presence of medium alone, 0.1 μg/ml, 1μg/ml, or 10 μg/ml IgG4 control, or 0.1 μg/ml, 1 μg/ml, or 10 μg/mlhuAb1 was performed substantially as described above. Table 9 belowshows the resuls of the multiplex analysis for all five synovial biopsyexplants tested for MMP-7 levels.

TABLE 7 Effect of huAb1 treatment on intact synovial biopsy cytokineproduction Analyte (pg/ml) per mg of Tissue: Mean (SEM), n = 4 0.1 μg/ml1 μg/ml 10 μg/ml 0.1 μg/ml 1 μg/ml 10 μg/ml Cytokine Medium IgG4 IgG4IgG4 huAb1 huAb1 huAb1 IL-1b 0.017 0.013 0.012 0.006 0.002 0.002 0.01(0.009) (0.011) (0.007) (0.005) (0.001) (0.001) (0.009) IL-6 240.680178.805 275.159 95.311 45.728 52.086 35.980 (146.459) (111.722)(107.706) (46.076) (18.001) (38.818) (9.206) IL-8 2237.972 831.6251375.193 610.201 244.370 180.821 298.966 (1594.031) (431.551) (311.382)(353.051) (62.781) (87.654) (188.495) CCL2/MPC1 1051.721 547.1901045.432 435.346 178.260 164.516 209.883 (646.236) (241.904) (218.350)(256.548) (42.634) (128.962) (103.918) CCL7/MCP3 8.883 6.427 12.1605.682 2.773 1.598 3.514 (4.843) (2.200) (4.515) (4.518) (2.054) (0.852)(2.864) CXCL5 80.589 30.389 70.981 67.473 11.212 10.875 33.917 (46.550)(13.460) (13.990) (55.635) (2.086) (5.341) (26.600) CXCL6 17.730 8.19110.017 4.083 3.779 1.588 1.105 (11.385) (4.189) (3.417) (1.310) (1.273)(0.893) (0.225) CXCL9/MIG 65.274 65.798 95.255 34.273 34.781 21.74824.291 (39.238) (27.685) (20.943) (19.210) (6.520) (14.365) (8.065)CXCL10/IP-10 55.609 40.343 48.644 15.895 14.615 12.951 16.019 (29.571)(21.332) (12.545) (11.744) (5.766) (7.450) (8.897) MMP2 532.546 407.589588.994 438.448 419.529 276.579 210.636 (298.570) (177.550) (218.276)(252.070) (136.061) (141.318) (77.979) MMP7 9.931 1.786 2.946 5.5880.518 0.273 24.226 (9.572) (1.298) (1.706) (3.566) (0.518) (0.273)(23.603) MMP9 92.909 191.612 290.114 135.964 119.953 98.286 148.330(46.738) (158.806) (209.547) (98.550) (88.836) (81.116) (109.991) TNFa0.215 0.104 0.256 0.074 0.033 0.016 0.018 (0.1411) (0.045) (0.111)(0.066) (0.018) (0.009) (0.015)

TABLE 8 Effect of huAb1 treatment on intact synovial biopsy MMP-2 andMMP-9 production. Analyte (pg/ml) per mg of Tissue: Mean (SEM), n = 6Cytokine Medium 0.1 μg/ml IgG4 1 μg/ml IgG4 10 μg/ml IgG4 0.1 μg/mlhuAb1 1 μg/ml huAb1 10 μg/ml huAb1 MMP2 736.3 (232.6) 666.3 (226.4)893.3 (289.1) 844.3 (306.0) 765.7 (254.8) 600.4 (243.2) 610.1 (350.9)MMP9 82.02 (31.52) 160.2 (103.6) 220.3 (140.1) 113.8 (65.06) 96.51(58.94) 83.02 (52.77) 143.4 (69.67)

TABLE 9 Effect of huAb1 treatment on intact synovial biopsy MMP-7production. Analyte (pg/ml) per mg of Tissue: Mean (SEM), n = 5 CytokineMedium 0.1 μg/ml IgG4 1 μg/ml IgG4 10 μg/ml IgG4 0.1 μg/ml huAb1 1 μg/mlhuAb1 10 μg/ml huAb1 MMP7 11.19 (7.52) 4.76 (3.13) 8.90 (6.10) 7.32(3.56) 4.61 (4.11) 3.02 (2.75) 27.09 (18.51)Table of Sequences

Table 10 provides certain sequences discussed herein. All polypeptideand antibody sequences are shown without leader sequences, unlessotherwise indicated.

TABLE 10 Sequences and Descriptions SEQ ID NO Description Sequence 1hCSF1R IPVIEPSVPE LVVKPGATVT LRCVGNGSVE WDGPPSPHWT LYSDGSSSIL(full-length, STNNATFQNT GTYRCTEPGD PLGGSAAIHL YVKDPARPWN VLAQEVVVFEno leader DQDALLPCLL TDPVLEAGVS LVRVRGRPLM RHTNYSFSPW HGFTIHRAKFsequence) IQSQDYQCSA LMGGRKVMSI SIRLKVQKVI PGPPALTLVP AELVRIRGEAAQIVCSASSV DVNFDVFLQH NNTKLAIPQQ SDFHNNRYQK VLTLNLDQVDFQHAGNYSCV ASNVQGKHST SMFFRVVESA YLNLSSEQNL IQEVTVGEGLNLKVMVEAYP GLQGFNWTYL GPFSDHQPEP KLANATTKDT YRHTFTLSLPRLKPSEAGRY SFLARNPGGW RALTFELTLR YPPEVSVIWT FINGSGTLLCAASGYPQPNV TWLQCSGHTD RCDEAQVLQV WDDPYPEVLS QEPFHKVTVQSLLTVETLEH NQTYECRAHN SVGSGSWAFI PISAGAHTHP PDEFLFTPVVVACMSIMALL LLLLLLLLYK YKQKPKYQVR WKIIESYEGN SYTFIDPTQLPYNEKWEFPR NNLQFGKTLG AGAFGKVVEA TAFGLGKEDA VLKVAVKMLKSTAHADEKEA LMSELKIMSH LGQHENIVNL LGACTHGGPV LVITEYCCYGDLLNFLRRKA EAMLGPSLSP GQDPEGGVDY KNIHLEKKYV RRDSGFSSQGVDTYVEMRPV STSSNDSFSE QDLDKEDGRP LELRDLLHFS SQVAQGMAFLASKNCIHRDV AARNVLLTNG HVAKIGDFGL ARDIMNDSNY IVKGNARLPVKWMAPESIFD CVYTVQSDVW SYGILLWEIF SLGLNPYPGI LVNSKFYKLVKDGYQMAQPA FAPKNIYSIM QACWALEPTH RPTFQQICSF LQEQAQEDRRERDYTNLPSS SRSGGSGSSS SELEEESSSE HLTCCEQGDI AQPLLQPNNY QFC 2 hCSF1RMGPGVLLLLL VATAWHGQGI PVIEPSVPEL VVKPGATVTL RCVGNGSVEW (full-length, +DGPPSPHWTL YSDGSSSILS TNNATFQNTG TYRCTEPGDP LGGSAAIHLY leaderVKDPARPWNV LAQEVVVFED QDALLPCLLT DPVLEAGVSL VRVRGRPLMR sequence)HTNYSFSPWH GFTIHRAKFI QSQDYQCSAL MGGRKVMSIS IRLKVQKVIPGPPALTLVPA ELVRIRGEAA QIVCSASSVD VNFDVFLQHN NTKLAIPQQSDFHNNRYQKV LTLNLDQVDF QHAGNYSCVA SNVQGKHSTS MFFRVVESAYLNLSSEQNLI QEVTVGEGLN LKVMVEAYPG LQGFNWTYLG PFSDHQPEPKLANATTKDTY RHTFTLSLPR LKPSEAGRYS FLARNPGGWR ALTFELTLRYPPEVSVIWTF INGSGTLLCA ASGYPQPNVT WLQCSGHTDR CDEAQVLQVWDDPYPEVLSQ EPFHKVTVQS LLTVETLEHN QTYECRAHNS VGSGSWAFIPISAGAHTHPP DEFLFTPVVV ACMSIMALLL LLLLLLLYKY KQKPKYQVRWKIIESYEGNS YTFIDPTQLP YNEKWEFPRN NLQFGKTLGA GAFGKVVEATAFGLGKEDAV LKVAVKMLKS TAHADEKEAL MSELKIMSHL GQHENIVNLLGACTHGGPVL VITEYCCYGD LLNFLRRKAE AMLGPSLSPG QDPEGGVDYKNIHLEKKYVR RDSGFSSQGV DTYVEMRPVS TSSNDSFSEQ DLDKEDGRPLELRDLLHFSS QVAQGMAFLA SKNCIHRDVA ARNVLLTNGH VAKIGDFGLARDIMNDSNYI VKGNARLPVK WMAPESIFDC VYTVQSDVWS YGILLWEIFSLGLNPYPGIL VNSKFYKLVK DGYQMAQPAF APKNIYSIMQ ACWALEPTHRPTFQQICSFL QEQAQEDRRE RDYTNLPSSS RSGGSGSSSS ELEEESSSEHLTCCEQGDIA QPLLQPNNYQ FC 5 hCSF1RIPVIEPSVPE LVVKPGATVT LRCVGNGSVE WDGPPSPHWT LYSDGSSSIL ECD.506STNNATFQNT GTYRCTEPGD PLGGSAAIHL YVKDPARPWN VLAQEVVVFEDQDALLPCLL TDPVLEAGVS LVRVRGRPLM RHTNYSFSPW HGFTIHRAKFIQSQDYQCSA LMGGRKVMSI SIRLKVQKVI PGPPALTLVP AELVRIRGEAAQIVCSASSV DVNFDVFLQH NNTKLAIPQQ SDFHNNRYQK VLTLNLDQVDFQHAGNYSCV ASNVQGKHST SMFFRVVESA YLNLSSEQNL IQEVTVGEGLNLKVMVEAYP GLQGFNWTYL GPFSDHQPEP KLANATTKDT YRHTFTLSLPRLKPSEAGRY SFLARNPGGW RALTFELTLR YPPEVSVIWT FINGSGTLLCAASGYPQPNV TWLQCSGHTD RCDEAQVLQV WDDPYPEVLS QEPFHKVTVQSLLTVETLEH NQTYECRAHN SVGSGSWAFI PISAGAH 6 hCSF1RIPVIEPSVPE LVVKPGATVT LRCVGNGSVE WDGPPSPHWT LYSDGSSSIL ECD.506-FcSTNNATFQNT GTYRCTEPGD PLGGSAAIHL YVKDPARPWN VLAQEVVVFEDQDALLPCLL TDPVLEAGVS LVRVRGRPLM RHTNYSFSPW HGFTIHRAKFIQSQDYQCSA LMGGRKVMSI SIRLKVQKVI PGPPALTLVP AELVRIRGEAAQIVCSASSV DVNFDVFLQH NNTKLAIPQQ SDFHNNRYQK VLTLNLDQVDFQHAGNYSCV ASNVQGKHST SMFFRVVESA YLNLSSEQNL IQEVTVGEGLNLKVMVEAYP GLQGFNWTYL GPFSDHQPEP KLANATTKDT YRHTFTLSLPRLKPSEAGRY SFLARNPGGW RALTFELTLR YPPEVSVIWT FINGSGTLLCAASGYPQPNV TWLQCSGHTD RCDEAQVLQV WDDPYPEVLS QEPFHKVTVQSLLTVETLEH NQTYECRAHN SVGSGSWAFI PISAGAHEPK SSDKTHTCPPCPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWYVDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKALPAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIAVEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVMHEALHNHYTQ KSLSLSPGK 7 cynoCSF1RMGPGVLLLLL VVTAWHGQGI PVIEPSGPEL VVKPGETVTL RCVGNGSVEW ECD (withDGPISPHWTL YSDGPSSVLT TTNATFQNTR TYRCTEPGDP LGGSAAIHLY leaderVKDPARPWNV LAKEVVVFED QDALLPCLLT DPVLEAGVSL VRLRGRPLLR sequence)HTNYSFSPWH GFTIHRAKFI QGQDYQCSAL MGSRKVMSIS IRLKVQKVIPGPPALTLVPA ELVRIRGEAA QIVCSASNID VDFDVFLQHN TTKLAIPQRSDFHDNRYQKV LTLSLGQVDF QHAGNYSCVA SNVQGKHSTS MFFRVVESAYLDLSSEQNLI QEVTVGEGLN LKVMVEAYPG LQGFNWTYLG PFSDHQPEPKLANATTKDTY RHTFTLSLPR LKPSEAGRYS FLARNPGGWR ALTFELTLRYPPEVSVIWTS INGSGTLLCA ASGYPQPNVT WLQCAGHTDR CDEAQVLQVWVDPHPEVLSQ EPFQKVTVQS LLTAETLEHN QTYECRAHNS VGSGSWAFIP ISAGAR 8cynoCSF1R MGPGVLLLLL VVTAWHGQGI PVIEPSGPEL VVKPGETVTL RCVGNGSVEW ECD FCDGPISPHWTL YSDGPSSVLT TTNATFQNTR TYRCTEPGDP LGGSAAIHLY (with leaderVKDPARPWNV LAKEVVVFED QDALLPCLLT DPVLEAGVSL VRLRGRPLLR sequence)HTNYSFSPWH GFTIHRAKFI QGQDYQCSAL MGSRKVMSIS IRLKVQKVIPGPPALTLVPA ELVRIRGEAA QIVCSASNID VDFDVFLQHN TTKLAIPQRSDFHDNRYQKV LTLSLGQVDF QHAGNYSCVA SNVQGKHSTS MFFRVVESAYLDLSSEQNLI QEVTVGEGLN LKVMVEAYPG LQGFNWTYLG PFSDHQPEPKLANATTKDTY RHTFTLSLPR LKPSEAGRYS FLARNPGGWR ALTFELTLRYPPEVSVIWTS INGSGTLLCA ASGYPQPNVT WLQCAGHTDR CDEAQVLQVWVDPHPEVLSQ EPFQKVTVQS LLTAETLEHN QTYECRAHNS VGSGSWAFIPISAGARGSEP KSSDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDELTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 3 Light chainMETDTLLLWV LLLWVPGSTG leader sequence 4 Heavy chain MAVLGLLLCL VTFPSCVLSleader sequence 9 Fab 0301EVQLQQSGPE LVRPGASVKM SCKASGYTFT DNYMIWVKQS HGKSLEWIGD heavy chainINPYNGGTTF NQKFKGKATL TVEKSSSTAY MQLNSLTSED SAVYYCARES variablePYFSNLYVMD YWGQGTSVTV SS region 10 Fab 0301NIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDNYMNWY QQKPGQPPKL light chainLIYAASNLES GIPARFSGSG SGTDFTLNIH PVEEEDAATY YCHLSNEDLS variableTFGGGTKLEI K region 11 Fab 0302EIQLQQSGPE LVKPGASVKM SCKASGYTFS DFNIHWVKQK PGQGLEWIGY heavy chainINPYTDVTVY NEKFKGKATL TSDRSSSTAY MDLSSLTSED SAVYYCASYF variableDGTFDYALDY WGQGTSITVS S region 12 Fab 0302DVVVTQTPAS LAVSLGQRAT ISCRASESVD NYGLSFMNWF QQKPGQPPKL light chainLIYTASNLES GIPARFSGGG SRTDFTLTID PVEADDAATY FCQQSKELPW variableTFGGGTRLEI K region 13 Fab 0311EIQLQQSGPD LMKPGASVKM SCKASGYIFT DYNMHWVKQN QGKSLEWMGE heavy chainINPNNGVVVY NQKFKGTTTL TVDKSSSTAY MDLHSLTSED SAVYYCTRAL variableYHSNFGWYFD SWGKGTTLTV SS region 14 Fab 0311DIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDSHMNWY QQKPGQPPKL light chainLIYTASNLES GIPARFSGSG SGADFTLTIH PVEEEDAATY YCQQGNEDPW variableTFGGGTRLEI K region 15 0301 heavy GYTFTDNYMI chain CDR1 16 0301 heavyDINPYNGGTT FNQKFKG chain CDR2 17 0301 heavy ESPYFSNLYV MDY chain CDR3 180301 light KASQSVDYDG DNYMN chain CDR1 19 0301 light AASNLES chain CDR220 0301 light HLSNEDLST chain CDR3 21 0302 heavy GYTFSDFNIH chain CDR122 0302 heavy YINPYTDVTV YNEKFKG chain CDR2 23 0302 heavy YFDGTFDYAL DYchain CDR3 24 0302 light RASESVDNYG LSFMN chain CDR1 25 0302 lightTASNLES chain CDR2 26 0302 light QQSKELPWT chain CDR3 27 0311 heavyGYIFTDYNMH chain CDR1 28 0311 heavy EINPNNGVVV YNQKFKG chain CDR2 290311 heavy ALYHSNFGWY FDS chain CDR3 30 0311 light KASQSVDYDG DSHMNchain CDR1 31 0311 light TASNLES chain CDR2 32 0311 light QQGNEDPWTchain CDR3 33 cAb 0301EVQLQQSGPE LVRPGASVKM SCKASGYTFT DNYMIWVKQS HGKSLEWIGD heavy chainINPYNGGTTF NQKFKGKATL TVEKSSSTAY MQLNSLTSED SAVYYCARESPYFSNLYVMD YWGQGTSVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 34 cAb 0301NIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDNYMNWY QQKPGQPPKL light chainLIYAASNLES GIPARFSGSG SGTDFTLNIH PVEEEDAATY YCHLSNEDLSTFGGGTKLEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 35 cAb 0302EIQLQQSGPE LVKPGASVKM SCKASGYTFS DFNIHWVKQK PGQGLEWIGY heavy chainINPYTDVTVY NEKFKGKATL TSDRSSSTAY MDLSSLTSED SAVYYCASYFDGTFDYALDY WGQGTSITVS SASTKGPSVF PLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTKTYTCNVDHKP SNTKVDKRVE SKYGPPCPPC RAPEFLGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNSTYRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVYTLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSR LTVDKSRWQE GNVFSCSVMH KALHNHYTQK SLSLSLGK 36 cAb 0302DVVVTQTPAS LAVSLGQRAT ISCRASESVD NYGLSFMNWF QQKPGQPPKL light chainLIYTASNLES GIPARFSGGG SRTDFTLTID PVEADDAATY FCQQSKELPWTFGGGTRLEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 37 cAb 0311EIQLQQSGPD LMKPGASVKM SCKASGYIFT DYNMHWVKQN QGKSLEWMGE heavy chainINPNNGVVVY NQKFKGTTTL TVDKSSSTAY MDLHSLTSED SAVYYCTRALYHSNFGWYFD SWGKGTTLTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 38 cAb 0311DIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDSHMNWY QQKPGQPPKL light chainLIYTASNLES GIPARFSGSG SGADFTLTIH PVEEEDAATY YCQQGNEDPWTFGGGTRLEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 39 h0301-H0QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD heavy chainINPYNGGTTF NQKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARES variablePYFSNLYVMD YWGQGTLVTV SS region 40 h0301-H1QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD heavy chainINPYNGGTTF NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCARES variablePYFSNLYVMD YWGQGTLVTV SS region 41 h0301-H2QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWIGD heavy chainINPYNGGTTF NQKFKGRATL TVDKSTSTAY MELSSLRSED TAVYYCARES variablePYFSNLYVMD YWGQGTLVTV SS region 42 H0302-H1QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWMGY heavy chainINPYTDVTVY NEKFKGRVTI TSDKSTSTAY MELSSLRSED TAVYYCASYF variableDGTFDYALDY WGQGTLVTVS S region 43 H0302-H2QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWIGY heavy chainINPYTDVTVY NEKFKGRATL TSDKSTSTAY MELSSLRSED TAVYYCASYF variableDGTFDYALDY WGQGTLVTVS S region 44 H0311-H1QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE heavy chainINPNNGVVVY NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCTRAL variableYHSNFGWYFD SWGQGTLVTV SS region 45 H0311-H2QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE heavy chainINPNNGVVVY NQKFKGTTTL TVDKSTSTAY MELSSLRSED TAVYYCTRAL variableYHSNFGWYFD SWGQGTLVTV SS region 46 h0301-L0EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL light chainLIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLS variableTFGGGTKVEI K region 47 h0301-L1NIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL light chainLIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLS variableTFGGGTKVEI K region 48 H0302-L0EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQSKELPW variableTFGQGTKVEI K region 49 H0302-L1EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPW variableTFGQGTKVEI K region 50 H0302-L2EIVVTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWF QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPW variableTFGQGTKVEI K region 51 H0311-L0EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQGNEDPW variableTFGQGTKVEI K region 52 H0311-L1DIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGADFTLTIS SLEPEDFAVY YCQQGNEDPW variableTFGQGTKVEI K region 53 h0301-H0QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD heavy chainINPYNGGTTF NQKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARESPYFSNLYVMD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 54 h0301-H1QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD heavy chainINPYNGGTTF NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCARESPYFSNLYVMD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 55 h0301-H2QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWIGD heavy chainINPYNGGTTF NQKFKGRATL TVDKSTSTAY MELSSLRSED TAVYYCARESPYFSNLYVMD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 56 H0302-H1QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWMGY heavy chainINPYTDVTVY NEKFKGRVTI TSDKSTSTAY MELSSLRSED TAVYYCASYFDGTFDYALDY WGQGTLVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTKTYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNSTYRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVYTLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSR LTVDKSRWQE GNVFSCSVMH KALHNHYTQK SLSLSLGK 57 H3002-H2QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWIGY heavy chainINPYTDVTVY NEKFKGRATL TSDKSTSTAY MELSSLRSED TAVYYCASYFDGTFDYALDY WGQGTLVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTKTYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNSTYRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVYTLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSR LTVDKSRWQE GNVFSCSVMH KALHNHYTQK SLSLSLGK 58 H0311-H1QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE heavy chainINPNNGVVVY NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCTRALYHSNFGWYFD SWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 59 H0311-H2QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE heavy chainINPNNGVVVY NQKFKGTTTL TVDKSTSTAY MELSSLRSED TAVYYCTRALYHSNFGWYFD SWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 60 h0301-L0EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL light chainLIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLSTFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC h0301-L1NIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL 61 light chainLIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLSTFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 62 H0302-L0EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQSKELPWTFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 63 H0302-L1EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPWTFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 64 H0302-L2EIVVTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWF QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPWTFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 65 H0311-L0EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQGNEDPWTFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 66 H0311-L1DIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGADFTLTIS SLEPEDFAVY YCQQGNEDPWTFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 67 HumanEEVSEYCSHM IGSGHLQSLQ RLIDSQMETS CQITFEFVDQ EQLKDPVCYL CSF1KKAFLLVQDI MEDTMRFRDN TPNAIAIVQL QELSLRLKSC FTKDYEEHDKACVRTFYETP LQLLEKVKNV FNETKNLLDK DWNIFSKNCN NSFAECSSQG HERQSEGS 68Human IL- NEPLEMWPLT QNEECTVTGF LRDKLQYRSR LQYMKHYFPI NYKISVPYEG 34VFRIANVTRL QRAQVSEREL RYLWVLVSLSATESVQDVLL EGHPSWKYLQEVQTLLLNVQ QGLTDVEVSP KVESVLSLLN APGPNLKLVR PKALLDNCFRVMELLYCSCC KQSSVLNWQD CEVPSPQSCS PEPSLQYAAT QLYPPPPWSPSSPPHSTGSV RPVRAQGEGL LP 69 Human QVQLVQSGAE VKKPGSSVKV SCKAS acceptor AFR1 70 Human WVRQAPGQGL EWMG acceptor A FR2 71 HumanRVTITADKST STAYMELSSL RSEDTAVYYC AR acceptor A FR3 72 Human WGQGTLVTVS Sacceptor A FR4 73 Human QVQLVQSGAE VKKPGSSVKV SCKAS acceptor B FR1 74Human WVRQAPGQGL EWMG acceptor B FR2 75 HumanRVTITADKST STAYMELSSL RSEDTAVYYC AR acceptor B FR3 76 Human WGQGTLVTVSSacceptor B FR4 77 Human QVQLVQSGAE VKKPGSSVKV SCKAS acceptor C FR1 78Human WVRQAPGQGL EWMG acceptor C FR2 79 HumanRVTITADKST STAYMELSSL RSEDTAVYYC AR acceptor C FR3 80 Human WGQGTLVTVS Sacceptor C FR4 81 Human EIVLTQSPAT LSLSPGERAT LSC acceptor D FR1 82Human WYQQKPGQAP RLLIY acceptor D FR2 83 HumanGIPARFSGSG SGTDFTLTIS SLEPEDFAVY YC acceptor D FR3 84 Human FGGGTKVEIKacceptor D FR4 85 Human EIVLTQSPAT LSLSPGERAT LSC acceptor E FR1 86Human WYQQKPGQAP RLLIY acceptor E FR2 87 HumanGIPARFSGSG SGTDFTLTIS SLEPEDFAVY YC acceptor E FR3 88 Human FGQGTKVEIKacceptor E FR4 89 Human EIVLTQSPAT LSLSPGERAT LSC acceptor F FR1 90Human WYQQKPGQAP RLLIY acceptor F FR2 91 HumanGIPARFSGSG SGTDFTLTIS SLEPEDFAVY YC acceptor F FR3 92 Human FGQGTKVEIKacceptor F FR4 93 mCSF1RAPVIEPSGPE LVVEPGETVT LRCVSNGSVE WDGPISPYWT LDPESPGSTL ECD FcTTRNATFKNT GTYRCTELED PMAGSTTIHL YVKDPAHSWN LLAQEVTVVEGQEAVLPCLI TDPALKDSVS LMREGGRQVL RKTVYFFSPW RGFIIRKAKVLDSNTYVCKT MVNGRESTST GIWLKVNRVH PEPPQIKLEP SKLVRIRGEAAQIVCSATNA EVGFNVILKR GDTKLEIPLN SDFQDNYYKK VRALSLNAVDFQDAGIYSCV ASNDVGTRTA TMNFQVVESA YLNLTSEQSL LQEVSVGDSLILTVHADAYP SIQHYNWTYL GPFFEDQRKL EFITQRAIYR YTFKLFLNRVKASEAGQYFL MAQNKAGWNN LTFELTLRYP PEVSVTWMPV NGSDVLFCDVSGYPQPSVTW MECRGHTDRC DEAQALQVWN DTHPEVLSQK PFDKVIIQSQLPIGTLKHNM TYFCKTHNSV GNSSQYFRAV SLGQSKQEPK SSDKTHTCPPCPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWYVDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKALPAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIAVEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVMHEALHNHYTQ KSLSLSPGK 94 HumanASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV IgG4 S241PHTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVESKYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEGNVFSCSVMHE ALHNHYTQKS LSLSLGK 95 HumanIgκRTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSGNSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC 96Human IL- VPPGEDSKDV AAPHRQPLTS SERIDKQIRY ILDGISALRK ETCNKSNMCE6; mature SSKEALAENN LNLPKMAEKD GCFQSGFNEE TCLVKIITGL LEFEVYLEYLQNRFESSEEQ ARAVQMSTKV LIQFLQKKAK NLDAITTPDP TTNASLLTKLQAQNQWLQDM TTHLILRSFK EFLQSSLRAL RQM 97 Human IL-APVRSLNCTL RDSQQKSLVM SGPYELKALH LQGQDMEQQV VFSMSFVQGE 1β; matureESNDKIPVAL GLKEKNLYLS CVLKDDKPTL QLESVDPKNY PKKKMEKRFVFNKIEINNKL EFESAQFPNW YISTSQAENM PVFLGGTKGG QDITDFTMQF VSS 98 Human IL-EGAVLPRSAK ELRCQCIKTY SKPFHPKFIK ELRVIESGPH CANTEIIVKL 8; matureSDGRELCLDP KENWVQRVVE KFLKRAENS 99 HumanQPDAINAPVT CCYNFTNRKI SVQRLASYRR ITSSKCPKEA VIFKTIVAKE CCL2;ICADPKQKWV QDSMDHLDKQ TQTPKT mature 100 HumanVPLSRTVRCT CISISNQPVN PRSLEKLEII PASQFCPRVE IIATMKKKGE CXCL10;KRCLNPESKA IKNLLKAVSK ERSKRSP mature 101 HumanVRSSSRTPSD KPVAHVVANP QAEGQLQWLN RRANALLANG VELRDNQLVV solublePSEGLYLIYS QVLFKGQGCP STHVLLTHTI SRIAVSYQTK VNLLSAIKSP TNF-αCQRETPEGAE AKPWYEPIYL GGVFQLEKGD RLSAEINRPD YLDFAESGQV YFGIIAL 102 HumanQPVGINTSTT CCYRFINKKI PKQRLESYRR TTSSHCPREA VIFKTKLDKE CCL7;ICADPTQKWV QDFMKHLDKK TQTPKL mature 103 HumanAGPAAAVLRE LRCVCLQTTQ GVHPKMISNL QVFAIGPQCS KVEVVASLKN CXCL5;GKEICLDPEA PFLKKVIQKI LDGGNKEN mature 104 HumanTPVVRKGRCS CISTNQGTIH LQSLKDLKQF APSPSCEKIE IIATLKNGVQ CXCL9;TCLNPDSADV KELIKKWEKQ VSQKKKQKNG KKHQKKKVLK VRKSQRSRQK mature KTT 105Human GPVSAVLTEL RCTCLRVTLR VNPKTIGKLQ VFPAGPQCSK VEVVASLKNG CXCL6;KQVCLDPEAP FLKKVIQKIL DSGNKKN mature 106 HumanYSLFPNSPKW TSKVVTYRIV SYTRDLPHIT VDRLVSKALN MWGKEIPLHF MMP-7;RKVVWGTADI MIGFARGAHG DSYPFDGPGN TLAHAFAPGT GLGGDAHFDE matureDERWTDGSSL GINFLYAATH ELGHSLGMGH SSDPNAVMYP TYGNGDPQNFKLSQDDIKGI QKLYGKRSNS RKK 107 HumanMRTPRCGVPD LGRFQTFEGD LKWHHHNITY WIQNYSEDLP RAVIDDAFAR MMP-9;AFALWSAVTP LTFTRVYSRD ADIVIQFGVA EHGDGYPFDG KDGLLAHAFP maturePGPGIQGDAH FDDDELWSLG KGVVVPTRFG NADGAACHFP FIFEGRSYSACTTDGRSDGL PWCSTTANYD TDDRFGFCPS ERLYTQDGNA DGKPCQFPFIFQGQSYSACT TDGRSDGYRW CATTANYDRD KLFGFCPTRA DSTVMGGNSAGELCVFPFTF LGKEYSTCTS EGRGDGRLWC ATTSNFDSDK KWGFCPDQGYSLFLVAAHEF GHALGLDHSS VPEALMYPMY RFTEGPPLHK DDVNGIRHLYGPRPEPEPRP PTTTTPQPTA PPTVCPTGPP TVHPSERPTA GPTGPPSAGPTGPPTAGPST ATTVPLSPVD DACNVNIFDA IAEIGNQLYL FKDGKYWRFSEGRGSRPQGP FLIADKWPAL PRKLDSVFEE RLSKKLFFFS GRQVWVYTGASVLGPRRLDK LGLGADVAQV TGALRSGRGK MLLFSGRRLW RFDVKAQMVDPRSASEVDRM FPGVPLDTHD VFQYREKAYF CQDRFYWRVS SRSELNQVDQ VGYVTYDILQ CPED108 Human APSPIIKFPG DVAPKTDKEL AVQYLNTFYG CPKESCNLFV LKDTLKKMQK MMP-2,FFGLPQTGDL DQNTIETMRK PRCGNPDVAN YNFFPRKPKW DKNQITYRII matureGYTPDLDPET VDDAFARAFQ VWSDVTPLRF SRIHDGEADI MINFGRWEHGDGYPFDGKDG LLAHAFAPGT GVGGDSHFDD DELWTLGEGQ VVRVKYGNADGEYCKFPFLF NGKEYNSCTD TGRSDGFLWC STTYNFEKDG KYGFCPHEALFTMGGNAEGQ PCKFPFRFQG TSYDSCTTEG RTDGYRWCGT TEDYDRDKKYGFCPETAMST VGGNSEGAPC VFPFTFLGNK YESCTSAGRS DGKMWCATTANYDDDRKWGF CPDQGYSLFL VAAHEFGHAM GLEHSQDPGA LMAPIYTYTKNFRLSQDDIK GIQELYGASP DIDLGTGPTP TLGPVTPEIC KQDIVFDGIAQIRGEIFFFK DRFIWRTVTP RDKPMGPLLV ATFWPELPEK IDAVYEAPQEEKAVFFAGNE YWIYSASTLE RGYPKPLTSL GLPPDVQRVD AAFNWSKNKKTYIFAGDKFW RYNEVKKKMD PGFPKLIADA WNAIPDNLDA VVDLQGGGHSYFFKGAYYLK LENQSLKSVK FGSIKSDWLG C

The invention claimed is:
 1. A method of reducing the level of at leastone factor selected from IL-6, IL-1β, IL-8, CCL2, CCL7, CXCL5, CXCL6,MMP-7, MMP-2, and MMP-9 in a subject with neoplasia, comprising (a)determining that the level of at least one factor selected from IL-6,IL-1β, IL-8, CCL2, CCL7, CXCL5, CXCL6, MMP-7, MMP-2, and MMP-9 iselevated in the subject, and (b) administering to the subject aneffective amount of an antibody to reduce the level of the at least onefactor selected from IL-6, IL-1β, IL-8, CCL2, CCL7, CXCL5, CXCL6, MMP-7,MMP-2, and MMP-9 in the subject, wherein the antibody binds human colonystimulating factor 1 receptor (CSF1R) and blocks binding of human colonystimulating factor 1 (CSF1) to human CSF1R and blocks binding of humanIL-34 to human CSF1R, and wherein the antibody comprises a heavy chaincomprising a heavy chain (HC) CDR1 having the sequence of SEQ ID NO: 15,an HC CDR2 having the sequence of SEQ ID NO: 16, and an HC CDR3 havingthe sequence of SEQ ID NO: 17, and a light chain comprising a lightchain (LC) CDR1 having the sequence of SEQ ID NO: 18, a LC CDR2 havingthe sequence of SEQ ID NO: 19, and a LC CDR3 having the sequence of SEQID NO:
 20. 2. The method of claim 1, further comprising (c) determiningthe level of the at least one factor selected from IL-6, IL-1β, IL-8,CCL2, CCL7, CXCL5, CXCL6, MMP-7, MMP-2, and MMP-9 in the subjectfollowing administration of the antibody to detect reduction of saidlevel.
 3. The method of claim 1, wherein the method comprises reducingthe level of at least one factor selected from IL-6 and IL-1β.
 4. Themethod of claim 3, wherein the method comprises reducing the level ofIL-6.
 5. The method of claim 1, wherein the antibody inhibitsligand-induced CSF1R phosphorylation in vitro.
 6. The method of claim 1,wherein the antibody is a humanized antibody.
 7. The method of claim 1,wherein the antibody is a Fab, an Fv, an scFv, a Fab′, or a (Fab′)₂. 8.The method of claim 1, wherein the method comprises administering atleast one additional therapeutic agent to the subject.
 9. The method ofclaim 1, wherein an elevated protein level of the at least one factorselected from IL-6, IL-1β, IL-8, CCL2, CCL7, CXCL5, CXCL6, MMP-7, MMP-2,and MMP-9 is determined.
 10. The method of claim 1, wherein an elevatedmRNA level of the at least one factor selected from IL-6, IL-1β, IL-8,CCL2, CCL7, CXCL5, CXCL6, MMP-7, MMP-2, and MMP-9 is determined.
 11. Themethod of claim 1, further comprising determining the level of CD16+monocytes in the subject before and/or after administration of theantibody.
 12. The method of claim 1, wherein the level of at least onefactor selected from IL-6, IL-1β, IL-8, CCL2, CCL7, CXCL5, CXCL6, MMP-7,MMP-2, and MMP-9 is determined in a blood sample from the subject. 13.The method of claim 1, wherein the antibody comprises a full-lengthheavy chain and a full-length light chain.
 14. The method of claim 13,wherein the antibody is an IgG antibody.
 15. The method of claim 14,wherein the antibody is an IgG4 antibody.
 16. The method of claim 15,wherein the IgG4 antibody comprises an S241P mutation.
 17. A method ofreducing the level of at least one factor selected from IL-6, IL-1β,IL-8, CCL2, CCL7, CXCL5, CXCL6, MMP-7, MMP-2, and MMP-9 in a subjectwith neoplasia, comprising (a) determining that the level of at leastone factor selected from IL-6, IL-1β, IL-8, CCL2, CCL7, CXCL5, CXCL6,MMP-7, MMP-2, and MMP-9 is elevated in the subject, and (b)administering to the subject an effective amount of an antibody toreduce the level of the at least one factor selected from IL-6, IL-1β,IL-8, CCL2, CCL7, CXCL5, CXCL6, MMP-7, MMP-2, and MMP-9 in the subject,wherein the antibody binds human colony stimulating factor 1 receptor(CSF1R) and blocks binding of human colony stimulating factor 1 (CSF1)to human CSF1R and blocks binding of human IL-34 to human CSF1R, andwherein the antibody comprises a heavy chain comprising the sequence ofSEQ ID NO: 39 and a light chain comprising the sequence of SEQ ID NO:46.
 18. The method of claim 17, wherein the antibody is a humanizedantibody.
 19. The method of claim 17, wherein the antibody is a Fab, anFv, an scFv, a Fab′, or a (Fab′)₂.
 20. The method of claim 17, whereinthe antibody is an IgG4 antibody comprising an S241P mutation.
 21. Themethod of claim 17, wherein the level of at least one factor selectedfrom IL-6, IL-1β, IL-8, CCL2, CCL7, CXCL5, CXCL6, MMP-7, MMP-2, andMMP-9 is determined in a blood sample from the subject.
 22. A method ofreducing the level of at least one factor selected from IL-6, IL-1β,IL-8, CCL2, CCL7, CXCL5, CXCL6, MMP-7, MMP-2, and MMP-9 in a subjectwith neoplasia comprising (a) determining that the level of at least onefactor selected from IL-6, IL-1β, IL-8, CCL2, CCL7, CXCL5, CXCL6, MMP-7,MMP-2, and MMP-9 is elevated in the subject, and (b) administering tothe subject an effective amount of an antibody to reduce the level ofthe at least one factor selected from IL-6, IL-1β, IL-8, CCL2, CXCL10,TNF-α, CCL7, CXCL5, CXCL9, CXCL6, MMP-7, MMP-2, and MMP-9 in thesubject, wherein the antibody binds human colony stimulating factor 1receptor (CSF1R) and blocks binding of human colony stimulating factor 1(CSF1) to human CSF1R and blocks binding of human IL-34 to human CSF1R,and wherein the antibody comprises a heavy chain comprising the sequenceof SEQ ID NO: 53 and a light chain comprising the sequence of SEQ ID NO:60.
 23. The method of claim 22, wherein the level of at least one factorselected from IL-6, IL-1β, IL-8, CCL2, CCL7, CXCL5, CXCL6, MMP-7, MMP-2,and MMP-9 is determined in a blood sample from the subject.